CDNA for human methylenetetrahydrofolate reductase

ABSTRACT

The present invention relates to a cDNA probe for human methylenetetrahydrofolate reductase (MTHFR), and its uses. The probe of the present invention may be used for the identification of sequence abnormalities in patients with severe or mild MTHFR deficiency, including cardiovascular patients and patients with neurologic symptoms. A human MTHFR protein which hybridizes to the probe of the present invention may be used for therapy of MTHFR-deficiency patients by biochemical or pharmacological approaches.

BACKGROUND OF THE INVENTION

(a) Field of the Invention

The invention relates to a cDNA probe for human methylenetetrahydrofolate reductase (MTHFR), and its uses.

(b) Description of Prior Art

Folic acid derivatives are coenzymes for several critical single-carbon transfer reactions, including reactions in the biosynthesis of purines, thymidylate and methionine. Methylenetetrahydrofolate reductase (MTHFR; EC 1.5.1.20) catalyzes the NADPH-linked reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, a co-substrate for methylation of homocysteine to methionine. The porcine liver enzyme, a flavoprotein, has been purified to homogeneity; it is a homodimer of 77-kDa subunits. Partial proteolysis of the porcine peptide has revealed two spatially distinct domains: an N-terminal domain of 40 kDa and a C-terminal domain of 37 kDa. The latter domain contains the binding site for the allosteric regulator S-adenosylmethionine.

Hereditary deficiency of MTHFR, an autosomal recessive disorder, is the most common inborn error of folic acid metabolism. A block in the production of methyltetrahydrofolate leads to elevated homocysteine with low to normal levels of methionine. Patients with severe deficiencies of MTHFR (0-20% activity in fibroblasts) can have variable phenotypes. Developmental delay, mental retardation, motor and gait abnormalities, peripheral neuropathy, seizures and psychiatric disturbances have been reported in this group, although at least one patient with severe MTHFR deficiency was asymptomatic. Pathologic changes in the severe form include the vascular changes that have been found in other conditions with elevated homocysteine, as well as reduced neurotransmitter and methionine levels in the CNS. A milder deficiency of MTHFR (35-50% activity) has been described in patients with coronary artery disease (see below). Genetic heterogeneity is likely, considering the diverse clinical features, the variable levels of enzyme activity, and the differential heat inactivation profiles of the reductase in patients' cells.

Coronary artery disease (CAD) accounts for 25% of deaths of Canadians. Cardiovascular risk factors (male sex, family history, smoking, hypertension, dyslipoproteinemia and diabetes) account for approximately 60 to 70% of our ability to discriminate CAD patients from healthy subjects. Elevated plasma homocysteine has also been shown to be an independent risk factor for cardiovascular disease.

Homocysteine is a sulfhydryl-containing amino acid that is formed by the demethylation of methionine. It is normally metabolized to cysteine (transsulfuration) or re-methylated to methionine. Inborn errors of metabolism (as in severe MTHFR deficiency) causing extreme elevations of homocysteine in plasma, with homocystinuria, are associated with premature vascular disease and widespread arterial and venous thrombotic phenomena. Milder elevations of plasma homocysteine (as in mild MTHFR deficiency) have been associated with the development of peripheral vascular disease, cerebrovascular disease and premature CAD.

Homocysteine remethylation to methionine requires the folic acid intermediate, 5-methyltetrahydrofolate, which is produced from 5,10-methylenetetrahydrofolate folate through the action of 5,10-methylenetetrahydrofolate reductase (MTHFR). Deficiency of MTHFR results in an inability to metabolize homocysteine to methionine; elevated plasma homocysteine and decreased methionine are the metabolic consequences of the block. Severe deficiencies of MTHFR (less than 20% of activity of controls) as described above, are associated with early-onset neurologic symptoms (mental retardation, peripheral neuropathy, seizures, etc.) and with atherosclerotic changes and thromboembolism. Milder deficiencies of MTHFR (35-50% of activity of controls), with a thermolabile form of the enzyme, are seen in patients with cardiovascular disease without obvious neurologic abnormalities.

In a survey of 212 patients with proven coronary artery disease, the thermolabile form of MTHFR was found in 17% of the CAD group and 5% of controls. In a subsequent report on 339 subjects who underwent coronary angiography, a correlation was found between thermolabile MTHFR and the degree of coronary artery stenosis. Again, traditional risk factors (age, sex, smoking, hypertension, etc.) were not significantly associated with thermolabile MTHFR. All the studies on MTHFR were performed by enzymatic assays of MTHFR in lymphocytes, with measurements of activity before and after heat treatment to determine thermolability of the enzyme.

Since 5-methyltetrahydrofolate, the product of the MTHFR reaction, is the primary form of circulatory folate, a deficiency in MTHFR might lead to other types of disorders. For example, periconceptual folate administration to women reduces the occurrence and recurrence of neural tube defects in their offspring. Neural tube defects are a group of developmental malformations (meningomyelocele, anencephaly, encephalocele) that arise due to failure of closure of the neural tube. Elevated levels of plasma homocysteine have been reported in mothers of children with neural tube defects. The elevated plasma homocysteine could be due to a deficiency of MTHFR, as described above for cardiovascular disease.

Neuroblastomas are tumors derived from neural crest cells. Many of these tumors have been reported to have deletions of human chromosome region lp36, the region of the genome to which MTHFR has been mapped. It is possible that MTHFR deletions/mutations are responsible for or contribute to the formation of this type of tumor. MTHFR abnormalities may also contribution to the formation of other types of tumors, such as colorectal tumors, since high dietary folate has been shown to be inversely associated with risk of colorectal adenomas.

MTHFR activity is required for homocysteine methylation to methionine. Methionine is necessary for the formation of S-adenosylmethionine, the primary methyl donor for methylation of DNA, proteins, lipids, neurotransmitters, etc. Abnormalities in MTHFR might lead to lower levels of methionine and S-adenosylmethionine, as well as to elevated homocysteine. Disruption of methylation processes could result in a wide variety of conditions, such as neoplasias, developmental anomalies, neurologic disorders, etc.

Although the MTHFR gene in Escherichia coli (metF) has been isolated and sequenced, molecular studies of the enzyme in higher organisms have been limited without the availability of a eukaryotic cDNA. In this communication, we report the isolation of a human cDNA for MTHFR, its chromosomal assignment, and the identification of mutations in MTHFR-deficient patients. This report represents the first molecular description of mutations in MTHFR deficiency.

It would be highly desirable to be provided with a cDNA probe for human methylenetetrahydrofolate reductase (MTHFR). This probe would be used for identification of sequence abnormalities in individuals with severe or mild MTHFR deficiency, including cardiovascular patients and patients with neurologic symptoms or tumors. The probe would also be used in gene therapy, isolation of the gene, and expression studies to produce the MTHFR protein. The probe would also provide the amino acid sequence of the human MTHFR protein, which would be useful for therapy of MTHFR deficiency by biochemical or pharmacological approaches.

It would be highly desirable to be provided with a molecular description of mutations in methylenetetrahydrofolate reductase deficiency.

SUMMARY OF THE INVENTION

One aim of the present invention is to provide a cDNA probe for human methylenetetrahydrofolate reductase (MTHFR).

Another aim of the present invention is to provide a molecular description of mutations in methylenetetrahydrofolate reductase deficiency.

Another aim of the present invention is to provide a nucleic acid and amino acid sequence for human methylenetetrahydrofolate reductase.

Another aim of the present invention is to provide potential therapy for individuals with methylenetetrahydrofolate reductase deficiency.

Another aim of the present invention is to provide a system for synthesis of MTHFR protein in vitro.

A further aim of the present invention is to provide for a technology/protocol for identification of sequence changes in the MTHFR gene.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A to 1F illustrate the first cDNA coding sequence (SEQ ID NO:1 and NO:2) for methylenetetrahydrofolate reductase (MTHFR);

FIG. 2 is the alignment of amino acids for human methylenetetrahydrofolate reductase (MTHFR), the metF genes from E. Coli (ECOMETF), and S. Typhimurium (STYMETF), and an unidentified open reading frame in Saccharomyces cerevisiae that is divergently transcribed from an excision repair gene (ysRAD1);

FIGS. 3A and 3B illustrate the sequencing and restriction enzyme analysis for the Arg to Ter substitution;

FIGS. 4A and 4B illustrate the sequencing and restriction enzyme analysis for the Arg to Gln substitution;

FIGS. 5A and 5B illustrates the sequence change and restriction enzyme analysis for the alanine to valine substitution;

FIGS. 6A-6C illustrates the total available sequence (SEQ ID NO:3 and NO:4) of human MTHFR cDNA;

FIGS. 7A and 7B illustrate the expression analysis of MTHFR cDNA in E. Coli, respectively (7A) the Western blot of bacterial extracts and tissues, and (7B) the thermolability assay of bacterial extracts;

FIGS. 8A to 8D illustrate the identification of a 5' splice site mutation leading to a 57 bp in-frame deletion of the cDNA;

FIGS. 9A to 9D illustrate the diagnostic restriction endonuclease analysis of 4 mutations;

FIGS. 10A to 10D illustrate the ASO hybridization analysis of 2 mutations; and

FIG. 11 illustrates the region of homology between human methylenetetrahydrofolate reductase (MTHFR) and human dihydrofolate reductase (DHFR).

DETAILED DESCRIPTION OF THE INVENTION

Sequencing of Peptides from Porcine MTHFR

Homogeneous native porcine MTHFR was digested with trypsin to generate a 40 kDa N-terminal fragment and a 31 kDa C-terminal fragment; the 31 kDa fragment is a proteolytic product of the 37 kDa fragment. The fragments were separated by SDS-PAGE, electroeluted, and the denatured fragments were digested with lysyl endopeptidase (LysC). The resulting peptides were separated by reversed-phase HPLC and subjected to sequence analysis by Edman degradation (details contained in Goyette P et al., Nature Genetics, 1994, 7:195-200).

Isolation and Sequencing of cDNAS

Two degenerate oligonucleotides were synthesized based on the sequence of a 30 amino acid porcine MTHFR peptide (first underlined peptide in FIG. 2). These were used to generate a 90 bp PCR product, encoding the predicted peptide, from reverse transcription-PCR reactions of 500 ng pig liver polyA+RNA. A pig-specific (non-degenerate, antisense) PCR primer was then synthesized from this short cDNA sequence. Using this primer and a primer for phage arms, a human liver λgt10 cDNA library (Clontech) was screened by PCR; this technique involved the generation of phage lysate stocks (50,000 pfu) which were boiled for 5 mins and then used directly in PCR reactions with these two primers. PCR fragments were then sequenced directly (Cycle Sequencing™ kit, GIBCO), and a positive clone was identified by comparison of the deduced amino acid sequence to the sequence of the pig peptide (allowing for inter-species variations). The positive stock was then replated at lower density and screened with the radiolabelled positive PCR product by plaque hybridization until a well-isolated plaque was identified. Phage DNA was purified and the insert was then subcloned into pBS+ (Bluescript) and sequenced on both strands (Cycle Sequencing™ kit, GIBCO and Sequenase™, Pharmacia). The deduced amino acid sequence of the human cDNA was aligned to the porcine peptide sequences, the metF genes from E.coli (ecometf, accession number VO1502) and S. typhimurium (stymetf, accession number XO7689) and with a previously unidentified open reading frame in Saccharomyces cerevisiae that is divergently transcribed with respect to the excision repair gene, ysRAD1 (accession number KO2070). The initial alignments were performed using BestFit™ in the GCG computer package, and these alignments were adjusted manually to maximize homologies.

In summary, degenerate oligonucleotide primers were designed to amplify a sequence corresponding to a 30-amino acid segment of a porcine peptide from the N-terminal region of the enzyme (first porcine peptide in FIG. 2). A 90-bp porcine cDNA fragment was obtained from reverse transcription/PCR of pig liver RNA. Sequencing of the PCR fragment confirmed its identity by comparison of the deduced amino acid sequence to the porcine peptide sequence. A nondegenerate oligonucleotide primer, based on the internal sequence of the porcine cDNA, was used in conjunction with primers for the phage arms to screen a human liver λgt10 cDNA library by PCR. The insert of the positive clone was isolated and sequenced. The sequence consisted of 1266 bp with one continuous open reading frame.

Homology with MTHFR in Other Species

The deduced amino acid sequence of the human cDNA was aligned with the metf genes from E.coli and S.typhimurium, as well as with a previously unidentified ORF in Saccharomyces cerevisiae that is divergently transcribed with respect to the excision repair gene, ysRAD1 (FIG. 2). The sequences homologous to 5 porcine peptides are underlined in FIG. 2. Three segments (residues 61-94, 219-240, and 337-351) correspond to internal peptide sequence from the N-terminal 40 kDa domain of the porcine liver enzyme. Residues 374-393 correspond to the upstream portion of the LysC peptide from the C-terminal domain of the porcine liver enzyme that is labeled when the enzyme is irradiated with UV light in the presence of (³ H-methyl)AdoMet; as predicted from the AdoMet labeling studies, this peptide lies at one end (N-terminal) of the 37 kDa domain. A fifth region of homology (residues 359-372) was also identified, but the localization of the porcine peptide within the native protein had not been previously determined.

Methylenetetrahydrofolate reductase (MTHFR) is an enzyme involved in amino acid metabolism, that is critical for maintaining an adequate methionine pool, as well as for ensuring that the homocysteine concentration does not reach toxic levels. The high degree of sequence conservation, from E.coli to Homo sapiens, attests to the significance of MTHFR in these species. The enzyme in E. coli (encoded by the metf locus) is a 33 kDa peptide that binds reduced FAD and catalyzes the reduction of methylenetetrahydrofolate to methyltetrahydrofolate. The metf enzyme differs from the mammalian enzyme in that it cannot be reduced by NADPH or NADH, and its activity is not allosterically regulated by S-adenosylmethionine. The native porcine enzyme is susceptible to tryptic cleavage between the N-terminal 40 kDa domain and the C-terminal 37 kDa domain, and this cleavage results in the loss of allosteric regulation by adenosylmethionine, but does not result in loss of catalytic activity. Since the homology between the bacterial and mammalian enzymes is within the N-terminal domain, this region must contain the flavin binding site and residues necessary to bind the folate substrate and catalyze its reduction. The domain structure of the human enzyme has not been elucidated, although the human enzyme has been reported to have a molecular mass of 150 kDa and is likely to be a homodimer of 77 kDa.

We predict that the point of cleavage between the two domains lies between residues 351 and 374 of the human sequence, based on the localization of peptides obtained from the isolated domains of the porcine enzyme. This region, containing the highly-charged sequence KRREED, is predicted to have the highest hydrophilicity and surface probability of any region in the deduced human sequence.

The N-terminus of the porcine protein has been sequenced, and the region encoding this part of the protein is missing from the human cDNA. We estimate that this cDNA is missing only a few residues at the N-terminus, since the predicted molecular mass of the deduced sequence upstream of the putative cleavage site (KRREED) is 40 kDa, and the measured molecular mass of the porcine N-terminal domain is also 40 kDa. When the bacterial, yeast and human sequences are aligned, the deduced human sequence contains an N-terminal extension of 40 amino acids; we suspect that this extension contains determinants for NADPH binding. Many pyridine nucleotide-dependent oxidoreductases contain such determinants at the N-terminus of the protein.

The C-terminus of the human sequence contains a peptide that is labeled when the protein is irradiated with ultraviolet light in the presence of tritiated AdoMet. The cDNA sequence we report here contains only about 7 kDa of the predicted 37 kDa mass of this domain, indicating that this cDNA is truncated at the 3' terminus as well. A number of peptides from the C-terminal porcine domain have also not been detected. As might be expected, given that the prokaryotic enzymes do not appear to be allosterically regulated by AdoMet, there are no significant homologies between the C-terminal region in this cDNA and the prokaryotic metF sequences. The alignment shown in FIG. 2 shows that the homologous sequences terminate just prior to the putative cleavage site of the human enzyme.

Chromosomal Assignment

In situ hybridization to metaphase human chromosomes was used for localization of the human gene. The analysis of the distribution of 200 silver grains revealed a significant clustering of grains 40 grains, in the p36.3-36.2 region of chromosome 1 (p<0.0001), with the majority of grains, 25 grains, observed over 1p36.3.

The isolation of the human cDNA has allowed us to localize the gene to chromosome lp36.3. The observation of one strong signal on that chromosome with little background is highly suggestive of a single locus with no pseudogenes. Southern blotting of human DNA revealed fragments of approximately 10 kb, predicting a gene of average size, since this cDNA encodes approximately half of the coding sequence.

Additional cDNA Sequences and Constructs for Expression Analysis

A human colon carcinoma cDNA library (gift of Dr. Nicole Beauchemin, McGill University) was screened by plaque hybridization with the original 1.3 kb cDNA to obtain additional coding sequences. A cDNA of 2.2 kb was isolated, which contained 1.3 kb of overlapping sequence to the original cDNA and 900 additional bp at the 3' end (FIG. 6). The amino acid sequence is identical to that of the original cDNA for the overlapping segment (codons 1-415) except for codon 177 (ASP) which was a GLY codon in the original cDNA. Analysis of 50 control chromosomes revealed an ASP codon at this position. The cDNA has an open reading frame of 1980 bp, 100 bp of 3' UTR and a poly A tail.

Sequencing was performed on both strands for the entire cDNA. Additional 5' sequences (800 bp) were obtained from a human kidney cDNA library (Clontech) but these sequences did not contain additional coding sequences and were therefore used for the PCR-based mutagenesis only (as described below) and not for the expression analysis. The two cDNAs (2.2 kb and 800 bp) were ligated using the EcoRI site at bp 199 and inserted into the Bluescrip™ vector (Stratagene). The 2.2 kb cDNA was subcloned into the expression. vector pTrc99A (Pharmacia) using the NcoI site at bp 11 and the XbaI site in the polylinker region of both the Bluescript™ and the pTrc99A vectors. Sequencing was performed across the cloning sites to verify the wild-type construct.

UTILITY OF INVENTION IN IDENTIFICATION OF MUTATIONS I. Identification of First Two Mutations in Severe MTHFR Deficiency

Total RNA of skin fibroblasts from MTHFR-deficient patients was reverse-transcribed and amplified by PCR for analysis by the single strand conformation polymorphism (SSCP) method (Orita, M. et al., Genomics, 1989, 5:8874-8879). Primers were designed to generate fragments of 250-300 bp and to cover the available cDNA sequences with small regions of overlap for each fragment at both ends. The first mutation identified by SSCP was a C to T substitution at bp 559 in patient 1554; this substitution converted an arginine codon to a termination codon (FIG. 3A). Since the mutation abolished a FokI site, restriction digestion was used for confirmation of the change and for screening additional patients for this mutation; a second patient (1627) was identified in this manner (FIG. 3B). The SSCP pattern for patient 1554 and the restriction digestion pattern for both patients was consistent with a homozygous mutant state or with a genetic compound consisting of the nonsense mutation with a second mutation that did not produce any detectable RNA (null allele). Studies in the parents are required for confirmation.

The second substitution (FIG. 4A) was a G to A transition at bp 482 in patient 1834 that converted an arginine into a glutamine residue. The substitution created a PstI site which was used to verify the substitution and to identify a second patient (1863) with this change (FIG. 4B). The SSCP analysis and the restriction digestion pattern were consistent with a heterozygous state for both patients. The arginine codon affected by this change is an evolutionarily-conserved residue, as shown in FIG. 2. This observation, in conjunction with the fact that the codon change is not conservative, makes a strong argument that the substitution is a pathologic change rather than a benign polymorphism. Furthermore, 35 controls (of similar ethnic background to that of the probands) were tested for this substitution by Southern blotting of PstI-digested DNA; all were negative.

The family of patient 1834 was studied. The symptomatic brother and the mother of the proband were all shown to carry this substitution, whereas the father was negative for the change (FIG. 4B). In the family of 1863, the mother of the proband was shown to be a carrier, while the father and an unaffected brother were negative.

Cell Lines

Cell line 1554 is from a Hopi male who was admitted at age three months with homocystinuria, seizures, dehydration, corneal clouding, hypotonia and candida sepsis. Folate distribution in cultured fibroblasts showed a Pediococcus cerivisiae/Lactobacillus casei(PC/LC) ratio of 0.52 (Control 0.14). There was no measurable methylenetetrahydrofolate reductase (MTHFR) activity (Control values=9.7 and 15.1 nmoles/h/mg protein; residual activity after treatment of control extracts at 55° C. for 20 min.=28% and 31%).

Cell line 1627 is from a Choctaw male who presented with poor feeding, apnea, failure to thrive, dehydration and homocystinuria at five weeks of age. He was subsequently found to have superior sagittal sinus thrombosis and hydrocephalus. The PC/LC ratio was 0.61 and the specific activity of MTHFR was 0.1 nmoles/h/mg protein. There is consanguinity in that the maternal and paternal grandmothers are thought to be "distantly related".

Cell line 1779 is from a French Canadian male with homocystinuria who first had limb weakness, incoordination, paresthesiae, and memory lapses at age 15 years, and was wheelchair-bound in his early twenties. His brother (cell line 1834) also has homocystinuria, but is 37 years old and asymptomatic. Specific activity of MTHFR was 0.7 and 0.9 nmole/h/mg protein for 1779 and 1834, respectively; the residual activity after heat treatment at 55° C. was 0.9% and 0% for 1779 and 1834, respectively.

Cell line 1863 is from a white male who was diagnosed at age 21 years because of a progressive gait disturbance, spasticity, cerebral white matter degeneration, and homocystinuria. He had a brother who died at age 21 years of neurodegenerative disease. Specific activity of MTHFR in fibroblast extracts was 1.76 nmoles/h/mg protein and the residual enzyme activity after treatment at 55° C. was 3.6%.

Mutation Analysis

Primers were designed from the cDNA sequence to generate 250-300 bp fragments which overlapped 50-75 bp at each end. The primer pairs were used in reverse transcription-PCR of 5 μg patient total fibroblast RNA. The PCR products were analyzed by a non-isotopic rapid SSCP protocol (PhastSystem™, Pharmacia), which uses direct silver staining for detection of single strands. Any PCR products from patients showing a shift on SSCP gels were purified by NuSieve (FMC Bioproducts) and sequenced directly (Cycle Sequencing™ kit, GIBCO) to identify the change. If the change affected a restriction site, then a PCR product was digested with the appropriate restriction endonuclease and analyzed on polyacrylamide gels. To screen for the Arg to Gln mutation in controls, 5 μg of PstI-digested DNA was run on 0.8% agarose gels and analyzed by Southern blotting using the radiolabelled cDNA by standard techniques.

II. Seven Additional Mutations at the Methylenetetrahydrofolate Reductase (MTHFR) Locus with Genotype: Phenotype Correlations in Severe MTHFR Deficiency

It is reported hereinbelow the characterization of 7 additional mutations at this locus: 6 missense mutations and a 5' splice site defect which activates a cryptic splice site in the coding sequence. We also present a preliminary analysis of the relationship between genotype and phenotype for all 9 mutations identified thus far at this locus. A nonsense mutation and 2 missense mutations (proline to leucine and threonine to methionine) in the homozygous state are associated with extremely low activity (0-3%) and onset of symptoms within the first year. Other missense mutations (arginine to cysteine and arginine to glutamine) are associated with higher enzyme activity and later onset of symptoms.

7 additional mutations at the MTHFR locus are described and the association between genotype, enzyme activity, and clinical phenotype in severe MTHFR deficiency is examined.

Patient Description

The clinical and laboratory findings of the patients have been reported in the published literature. Residual MTHFR activity was previously measured in cultured fibroblasts at confluence.

Patient 354, an African-American girl, was diagnosed at age 13 years with mild mental retardation. Her sister, patient 355 was diagnosed at age 15 years with anorexia, tremor, hallucinations and progressive withdrawal. In patient 354, residual MTHFR activity was 19% and in her sister, 355, it was 14% of control values. The residual activity after heating had equivalent thermal stability to control enzyme.

Patient 1807, a Japanese girl whose parents are first cousins, had delayed walking and speech until age 2 years, seizures at age 6 years and a gait disturbance with peripheral neuropathy at age 16 years. Residual activity of MTHFR was 3% and the enzyme was thermolabile.

Patient 735, an African-Indian girl, was diagnosed at age 7 months with microcephaly, progressive deterioration of mental development, apnea and coma. Residual activity of MTHFR was 2% of control levels. Thermal properties were not determined.

Patient 1084, a Caucasian male, was diagnosed at age 3 months with an infantile fibrosarcoma. He was found to be hypotonic and became apneic. He died at the age of 4 months. Residual activity of MTHFR was not detectable. Thermal properties were not determined.

Patient 356, the first patient reported with MTHFR deficiency, is an Italian-American male who presented at age 16 years with muscle weakness, abnormal gait and flinging movements of the upper extremities. MTHFR residual activity was 20% of control values; activity was rapidly and exponentially inactivated at 55°.

Patient 458, a Caucasian male, was diagnosed at age 12 years with ataxia and marginal school performance. Residual MTHFR activity was approximately 10%, and the activity was thermolabile.

Patient 1396, a Caucasian female, was described as clumsy and as having a global learning disorder in childhood. At age 14 years, she developed ataxia, foot drop, and inability to walk. She developed deep vein thrombosis and bilateral pulmonary emboli. Residual activity of MTHFR was 14% and the enzyme was thermolabile.

Genomic Structure and Intronic Primers Exon nomenclature is based on available cDNA sequence in Goyette et al. (Nature Genetics, 1994, 7:195-200). Exon 1 has been arbitrarily designated as the region of cDNA from bp 1 to the first intron. Identification of introns was performed by amplification of genomic DNA using cDNA primer sequences. PCR products that were greater in size than expected cDNA sizes were sequenced directly.

Mutation Detection

Specific exons (see Table 1 for primer sequences) were amplified by PCR from genomic DNA and analyzed by the SSCP protocol. SSCP was performed with the Phastgel™ system (Pharmacia), a non-isotopic rapid SSCP protocol, as previously described (Goyette P et al., Nature Genetics, 1994, 7:195-200), or with ³⁵ S-labeled PCR products run on 6% acrylamide: 10% glycerol gels at room temperature (6 watts, over-night). In some cases, the use of restriction endonucleases, to cleave the PCR product before SSCP analysis, enhanced the detection of band shifts. PCR fragments with altered mobility were sequenced directly (GIBCO, Cycle Sequencing™ kit). If the sequence change affected a restriction endonuclease site, then the PCR product was digested with the appropriate enzyme and analyzed by PAGE. Otherwise, allele-specific oligonucleotide (ASO) hybridization was performed on a dot blot of the PCR-amplified exon.

                                      TABLE 1                                      __________________________________________________________________________     PCR Primers for DNA amplification and mutation                                  analysis of MTHFR                                                                                                    Fragment                                                                        Exon Primer Type Primer Sequence                                              (5'→3') Location Size            __________________________________________________________________________                                            (bp)                                    1  Sense  AGCCTCAACCCCTGCTTGGAGG (SEQ ID NO:5)                                                                   C    271                                        Antisense TGACAGTTTGCTCCCCAGGCAC (SEO ID NO:6) I                              4 Sense TGAAGGAGAAGGTGTCTGCGGGA (SEQ ID NO:7) C 198                             Antisense AGGACGGTGCGGTGAGAGTGG (SEQ ID NO:8) I                               5 Sense CACTGTGGTTGGCATGGATGATG (SEQ ID NO:9) I 292                             Antisense GGCTGCTCTTGGACCCTCCTC (SEQ ID NO:10) I                              6 Sense TGCTTCCGGCTCCCTCTAGCC (SEQ ID NO:11) I 251                              Antisense CCTCCCGCTCCCAAGAACAAAG (SEQ ID NO:12) I                           __________________________________________________________________________

                                      TABLE 2                                      __________________________________________________________________________     Summary of genotypes, enzyme activity, age at onset,                             and background of patients with MTHFR deficiency                                          Amino acid %                                                        Patient.sup.a BPChanges.sup.b changes Activity Age at Onset Background       __________________________________________________________________________     1907 C784T/C784T                                                                            Pro→Leu/Pro→Leu                                                             3    within 1st year                                                                        Japanese                                    735 C9927/C692T Thr→Met/Thr→Met 2 7 months African Indian        1084 C692T/C692T Thr→M/Thr→Met 0 3 months Caucasian                                                  1554 CS59T/C559T Arg→Ter/Arg.                                          fwdarw.Ter 0 1 month Native American             (Hopl)                                                                    1627 C559T/C559T Arg→Ter/Arg→Ter 1 1 month Native                                                   American                                         (Choctaw)                                                                 356 C965T/C965T Arg→Cys/Arg→Cys 20 16 yrs Italian                                                   American                                    458 C1015T/G167A Arg→Cys/Arg→Gln 10 11 yrs Caucasian                                                 1396 C1061T/G167A Arg→Cys/Arg                                          →Gln 14 14 yrs Caucasian                                                 1779.sup.c G482A/7 Arg→Gln/7                                           8 15 yrs French Canadian                    1834.sup.c G482A/7 Arg→Gln/7 7 Asymptomatic French Canadian                                                     37 yrs                                 1863 G482A/7 Arg→Gln/7 14 21 yrs Caucasian                              354.sup.d 792 + 1G→A/7 5'splice site/7 19 13 yrs African                                                   American                                    355.sup.d 792 + 1G→A/7 5'splice site/7 14 11 yrs African              __________________________________________________________________________                                          American                                   .sup.a Patients 1554 1627, 1779, 1834 and 1863 were previously reported b      Goyette et al. (1994).                                                         .sup.b 7 = unidentified mutation.                                              .sup.c Patients 1779 and 1634 are sibs.                                        .sup.d Patients 354 and 355 are sibs.                                    

(1) 5' Splice Site Mutation

Amplification of cDNA, bp 653-939, from reverse-transcribed total fibroblast RNA revealed 2 bands in sisters 354 and 355: a smaller PCR fragment (230 bp) in addition to the normal 287 bp allele (FIG. 8A). FIG. 8A is the PAGE analysis of amplification products of cDNA bp 653-939, from reverse transcribed RNA. Controls have the expected 287 bp fragment while patients 354 and 355 have an additional 230 bp fragment. Sequencing of the smaller fragment identified a 57 bp in-frame deletion which would remove 19 amino acids (FIG. 8B). FIG. 8B is the direct sequencing of the PCR products from patient 354. The 57 bp deletion spans bp 736-792 of the cDNA. An almost perfect 5' splice site (boxed) is seen at the 5' deletion breakpoint. Analysis of the sequence at the 5' deletion breakpoint in the undeleted fragment revealed an almost perfect 5' splice site consensus sequence (AG/gcatgc). This observation suggested the presence of a splicing mutation in the natural 5' splice site that might activate this cryptic site, to generate the deleted allele. The sequence following the deletion breakpoint, in the mutant allele, corresponded exactly to the sequence of the next exon. Amplification of genomic DNA, using the same amplification primers as those used for reverse-transcribed RNA, generated a 1.2 kb PCR product indicating the presence of an intron. Direct sequencing of this PCR fragment in patient 354 identified a heterozygous G→A substitution in the conserved GT dinucleotide of the intron at the 5' splice site (FIG. 8C). FIG. 8C is the sequencing of the 5' splice site in control and patient 354. The patient carries a heterozygous G→A substitution in the 5' splice site (boxed). Intronic sequences are in lower case. This substitution abolished a HphI restriction endonuclease site which was used to confirm the mutation in the 2 sisters (FIG. 8D). FIG. 8D is the HphI restriction endonuclease analysis on PCR products of DNA for exon 4 of patients 354 and 355, and of 3 controls (C). The 198 bp PCR product has 2 HphI sites. The products of digestion for the control allele are 151, 24 and 23 bp. The products of digestion for the mutant allele are 175 and 23 bp due to the loss of a HphI site. The fragments of 24 and 23 bp have been run off the gel.

(2) Patients with Homozygous Coding Substitutions

SSCP analysis of exon 4 for patient 1807 revealed an abnormally-migrating fragment, which was directly sequenced to reveal a homozygous C→T substitution (bp 764) converting a proline to a leucine residue. This change creates a MnlI restriction endonuclease site, which was used to confirm the homozygous state of the mutation (FIG. 9A). FIG. 9A is the MnlI restriction analysis of exon 4 PCR products for patient 1807 and 3 controls (C). Expected fragments: control allele, 90, 46, 44, 18 bp; mutant allele, 73, 46, 44, 18, 17 bp. An additional band at the bottom of the gel is the primer. Fifty independent control Caucasian chromosomes and 12 control Japanese chromosomes were tested by restriction analysis; all were negative for this mutation. Homozygosity in this patient is probably due to the consanguinity of the parents.

Patients 735 and 1084 had the same mutation in exon 4, in a homozygous state: a C→T substitution (bp 692) which converted an evolutionarily-conserved threonine residue to a methionine residue, and abolished a NlaIII restriction endonuclease site. Allele-specific oligonucleotide hybridization to amplified exon 4 (FIGS. 10A and 10B) was used to confirm the mutation in these 2 patients and to screen 60 independent chromosomes, all of which turned out to be negative. FIG. 10A is the hybridization of mutant oligonucleotide (692T) to exon 4 PCR products from patients 735, 1084 and 30 controls. Only DNA from patients 735 and 1084 hybridized to this probe. FIG. 10B is the hybridization of normal oligonucleotide (692C) to stripped dot blot from A. All control DNAs hybridized to this probe.

Patient 356 showed a shift on SSCP analysis of exon 5. Direct sequencing revealed a homozygous C→T substitution (bp 985) which converted an evolutionarily-conserved arginine residue to cysteine; the substitution abolished an AciI restriction endonuclease site. This was used to confirm the homozygous state of the mutation in patient 356 (FIG. 9B) and its presence in the heterozygous state in both parents. Fifty independent control chromosomes, tested in the same manner, were negative for this mutation. FIG. 9B is the AciI restriction analysis of exon 5 PCR products for patient 356, his father (F), his mother (M), and 3 controls (C). Expected fragments: control allele, 129, 105, 90, 68 bp; mutant allele, 195, 129, 68 bp.

(3) Patients Who are Genetic Compounds

Patient 458 is a compound heterozygote of a mutation in exon 5 and a mutation in exon 1. The exon 5 substitution (C→T at bp 1015) resulted in the substitution of a cysteine residue for an arginine residue; this abolished a HhaI restriction endonuclease site, which was used to confirm the mutation in patient 458 (FIG. 9C) and to show that 50 control chromosomes were negative. FIG. 9C is the HhaI restriction analysis of exon 5 PCR products for patient 458 and 4 controls (C). Expected fragments: control allele, 317 and 75 bp; mutant allele 392 bp. The 75 bp fragment is not shown in FIG. 9C. The second mutation was a heterozygous G→A substitution (bp 167) converting an arginine to a glutamine residue. Allele-specific oligonucleotide hybridization to amplified exon 1 confirmed the heterozygous state of this mutation in patient 458 and identified a second patient (1396) carrying this mutation also in the heterozygous state (FIGS. 10C and 10D). FIG. 10C is the hybridization of mutant oligonucleotide (167A) to exon 1 PCR products from patients 458, 1396 and 31 controls. FIG. 10D is the hybridization of normal oligonucleotide (167G) to stripped dot blot from C. None of the 62 control chromosomes carried this mutation.

The second mutation in patient 1396 was identified in exon 6: a heterozygous C→T substitution (bp 1081) that converted an arginine residue to a cysteine residue, and abolished a HhaI restriction endonuclease site. Restriction analysis confirmed the heterozygous substitution in 1396 (FIG. 9D) and showed that 50 control chromosomes were negative. FIG. 9D is the HhaI restriction analysis of exon 6 PCR products for patient 1396 and 2 controls (C). Expected fragments: control allele, 152, 86, 13 bp; mutant allele 165, 86 bp. The 13 bp fragment has been run off the gel.

(4) Additional Sequence Changes

HhaI analysis of exon 6, mentioned above, revealed a DNA polymorphism. This change is a T→C substitution at bp 1068 which does not alter the amino acid (serine), but creates a HhaI recognition site. T at bp 1068 was found in 9% of tested chromosomes. Sequence analysis identified 2 discrepancies with the published cDNA sequence: a G→A substitution at bp 542 which converts the glycine to an aspartate codon, and a C→T change at bp 1032 which does not alter the amino acid (threonine). Since all DNAs tested (>50 chromosomes) carried the A at bp 542 and the T at bp 1032, it is likely that the sequence of the original cDNA contained some cloning artifacts.

Genotype:Phenotype Correlation

Table 2 summarizes the current status of mutations in severe MTHFR deficiency. In 8 patients, both mutations have been identified; in 5 patients (3 families), only 1 mutation has been identified. Overall the correlation between the genotype, enzyme activity, and phenotype is quite consistent. Five patients, with onset of symptoms within the first year of life, had ≦3% of control activity. Three of these patients had missense mutations in the homozygous state: two patients with the threonine to methionine substitution (C692T) and one patient with the proline to leucine substitution (C764T). The nonsense mutation (C559T) in the homozygous state in patients 1554 and 1627 (previously reported in Goyette P et al., Nature Genetics, 1994, 7:195-200) is also associated with a neonatal severe form, as expected.

The other patients in Table 2 had ≧6% of control activity and onset of symptoms within or after the 2nd decade of life; the only exception is patient 1834, as previously reported (Goyette P et al., Nature Genetics, 1994, 7:195-200). The three patients (356, 458 and 1396) with missense mutations (G167A, C985T, C1015T and C1081T) are similar to those previously reported (patients 1779, 1834 and 1863) who had an arginine to glutamine substitution and a second unidentified mutation (Goyette P et al., Nature Genetics, 1994, 7:195-200). The sisters with the 5' splice mutation and an unidentified second mutation also had levels of activity in the same range and onset of symptoms in the second decade, but the activity is likely due to the second unidentified allele.

Discussion

The patients come from diverse ethnic backgrounds. Although patients 1554 and 1627 are both Native Americans, the mutations occur on different haplotypes, suggesting recurrent mutation rather than identity by descent. Since the substitution occurs in a CpG dinucleotide, a "hot spot" for mutation, recurrent mutation is a reasonable hypothesis. It is difficult to assess whether some mutations are population-specific since the numbers are too small at the present time.

MTHFR deficiency is the most common inborn error of folate metabolism, and a major cause of hereditary homocysteinemia. The recent isolation of a cDNA for MTHFR has permitted mutational analysis at this locus, with the aims of defining important domains for the enzyme and of correlating genotype with phenotype in MTHFR-deficient patients.

Our definition of a disease-causing substitution, as distinct from a benign polymorphism, is based on 3 factors: (1) absence of the change in at least 50 independent control chromosomes; (2) presence of the amino acid in the bacterial enzyme, attesting to its evolutionary significance and (3) whether the change in amino acid is conservative. Although expression of the substitutions is required to formally prove that they are not benign, the criteria above allow us to postulate that the changes described in this report are likely to affect activity.

The 7 mutations described here (6 single amino acid substitutions and a 5' splice site mutation) bring the total to 9 mutations identified thus far in severe MTHFR deficiency and complete the mutation analysis for 8 patients. The identification of each mutation in only one or two families points to the striking degree of genetic heterogeneity at this locus. Seven of the 9 mutations are located in CpG dinucleotides, which are prone to mutational events.

5' Splice Site Mutation

The G→A substitution at the GT dinucleotide of the 5' splice site in patients 354 and 355 results in a 57 bp in-frame deletion of the coding sequence, which should delete 19 amino acids of the protein. This deletion occurs as a result of the activation of a cryptic 5' splice site (AG/gc) even though this cryptic site does not have a perfect 5' splice site consensus sequence (AG/gt). However, GC (instead of GT) as the first 2 nucleotides of an intron have been reported in several naturally-occurring splice sites, such as in the genes for human prothrombin and human adenine phosphoribosyltransferase and twice within the gene for the largest subunit of mouse RNA polymerase II. The remaining nucleotides of the cryptic site conform to a normal splice site consensus sequence with its expected variations (A₆₀ G₇₉ /g₁₀₀ t₁₀₀ a₅₉ a₇₁ g₈₂ t₅₀). It is unlikely that the deleted enzyme resulting from this aberrantly-spliced mRNA would have any activity; 8 of the 19 deleted amino acids are conserved in the E. coli enzyme. Although the 2 patients show residual enzyme activity in the range of 20% of controls, the activity is probably due to the unidentified second allele in these patients.

6 Missense Mutations

The Arg→Cys substitution (C1081T) in patient 1396 is within a hydrophilic sequence previously postulated to be the linker region between the catalytic and regulatory domains of MTHFR (Goyette P et al., Nature Genetics, 1994, 7:195-200). These 2 domains are readily separable by mild trypsinization of the porcine enzyme. The linker domain, a highly-charged region, is likely to be located on the outside surface of the protein and therefore more accessible to proteolysis. Because the Arg→Cys substitution converts a charged hydrophilic residue to an uncharged polar residue, it cannot be considered a conservative change, and could affect the stability of the enzyme.

The 2 Arg→Cys substitutions identified in patients 356 and 458 (C985T and C1015T, respectively) may be involved in binding the FAD cofactor. Previous work in the literature showed that heating fibroblast extracts at 55°, in the absence of the FAD cofactor, inactivated MTHFR completely. The addition of FAD to the reaction mixture before heat inactivation restored some enzyme activity to control extracts and to extracts from some patients, while the extracts of patients 356 and 458 were unaffected. Based on these observations, it was suggested that these 2 patients had mutations affecting a region of the protein involved in binding FAD. The 2 mutations are found in close proximity to each other, within 11 amino acids. In patient 356, the Arg residue is evolutionarily-conserved in E. coli and is found in a stretch of 9 conserved amino acids, suggesting a critical role for this residue; the altered Arg residue in patient 458 is not evolutionarily-conserved. Crystal structure analysis of medium chain acyl-CoA dehydrogenase (MCAD), a flavoprotein, has defined critical residues involved in the binding of FAD. Two consecutive residues of the MCAD protein, Met165 and Trp166, involved in interactions with FAD, can also be identified in MTHFR, 3 and 4 amino acids downstream, respectively, from the Arg residue altered in patient 458.

The Thr→Met substitution (C692T), is found in a region of high conservation with the E. coli enzyme and in a region of good homology with human dihydrofolate reductase (DHFR) (FIG. 11). In FIG. 11,=is identity;  is homology; and ⋄ is identity to bovine DHFR enzyme. An asterisk (*) indicates location of Thr→Met substitution. Considering the early-onset phenotype of the patients, one can assume that the threonine residue is critical for activity or that it contributes to an important domain of the protein. This region of homology in DHFR contains a residue, Thr136, which has been reported to be involved in folate binding. This Thr residue in DHFR aligns with a Ser residue in MTHFR, an amino acid with similar biochemical properties. The Thr→Met substitution is located 8 amino acids downstream from this Ser codon, in the center of the region of homology between the 2 enzymes. We therefore hypothesize that the Thr→Met substitution may alter the binding of the folate substrate.

The G167A (Arg→Gln) and C764T (Pro→Leu) substitutions both affect non-conserved amino acids. Their importance in the development of MTHFR deficiency cannot be determined at the present time. All the mutations identified thus far are located in the 5' end of the coding sequence, the region thought to encode the catalytic domain of MTHFR. Mutation analysis has been useful in beginning to address the structure: function properties of the enzyme as well as to understand the diverse phenotypes in severe MTHFR deficiency.

III. Identification of A→V Mutation

SSCP analysis and direct sequencing of PCR fragments were used to identify a C to T substitution at bp 677, which converts an alanine residue to a valine residue (FIG. 5A). The primers for analysis of the A→V change are: 5'-TGAAGGAGAA GGTGTCTGCG GGA-3' (SEQ ID NO:13) (exonic) and 5'-AGGACGGTGC GGTGAGAGTG-3' (SEQ ID NO:14) (intronic); these primers generate a fragment of 198 bp. FIG. 5A depicts the sequence of two individuals, a homozygote for the alanine residue and a homozygote for the valine residue. The antisense strands are depicted. This alteration creates a HinfI site (FIG. 5B), which was used to screen 114 unselected French Canadian chromosomes; the allele frequency of the substitution was 0.38. The substitution creates a HinfI recognition sequence which digests the 198 bp fragment into a 175 bp and a 23 bp fragment; the latter fragment has been run off the gel. FIG. 5B depicts the three possible genotypes. The frequency of the 3 genotypes were as follows: -/-, 37%; +/-, 51%; and +/+, 12% (the (+) indicates the presence of the HinfI restriction site and a valine residue).

The alanine residue is conserved in porcine MTHFR, as well as in the corresponding metF and stymetF genes of E. Coli and S. Typhimurium, respectively. The strong degree of conservation of this residue, and its location in a region of high homology with the bacterial enzymes, alluded to its importance in enzyme structure or function. Furthermore, the frequency of the (+/+) genotype was consistent with the frequency of the thermolabile MTHFR variant implicated in vascular disease.

Clinical Material

To determine the frequency of the A→V mutation, DNA from 57 individuals from Quebec was analyzed by PCR and restriction digestion. The individuals, who were all French Canadian, were not examined clinically or biochemically.

The 40 individuals analyzed in Table 3 had been previously described in Engbersen et al. (Am. J. Hum. Genet., 1995, 56:142-150). Of the 13 cardiovascular patients, 8 had cerebrovascular arteriosclerosis and 5 had peripheral arteriosclerosis. Five had thermolabile MTHFR while 8 had thermostable MTHFR (greater than 33% residual activity after heating). Controls and patients were all Dutch-Caucasian, between 20-60 years of age. None of these individuals used vitamins which could alter homocysteine levels. Enzyme assays and homocysteine determinations were also reported by Engbersen et al. (Am. J. Hum. Genet., 1995, 56:142-150).

                  TABLE 3                                                          ______________________________________                                         Correlation between MTHFR genotype and enzyme activity,                          thermolability and plasma homocysteine level                                               -/-       +/-       +/+                                            n = 19 n = 9 n = 12                                                          ______________________________________                                         specific activity.sup.a,b                                                                    22.9 ± 1.7                                                                            15.0 ± 0.8                                                                             6.9 ± 0.6                                  (nmol CH.sub.2 O/mg.protein/hr) (11.8-33.8)  (10.2-18.8)  (2.6-10.2)                                            residual activity after 66.8 ± 1.5                                         56.2 ± 2.8 21.8 ± 2.8                    heating.sup.a,b (%) (55-76)  (41-67)  (10-35)                                  plasma homocysteine.sup.a,c 12.6 ± 1.1  13.8 ± 1.0 22.4 ± 2.9                                          (μM)(after fasting)  (7-21)                                                (9.6-20)  (9.6-42)                             plasma homocysteine.sup.a,c 41.3 ± 5.0.sup.d     41 ± 2.8  72.6                                          ± 11.7.sup.e                                (μM)(post-methionine load) (20.9-110)  (29.1-54)   (24.4-159)             ______________________________________                                          .sup.a oneway anova p < .01                                                    .sup.b paired t test for all combinations p < .01                              .sup.c paired t test p < .05 for +/+ group versus +/- group or -/- group;      p > .05 for +/- versus -/- group.                                              .sup.d n = 18 for this parameter                                               .sup.e n = 11 for this parameter                                         

Enzyme activity and plasma homocysteine were determined as previously reported. Each value represents mean ± standard error. The range is given in parentheses below the mean.

Correlation of A→V Mutation with Altered MTHFR Function

A genotypic analysis was performed and enzyme activity and thermolability were measured in a total of 40 lymphocyte pellets from patients with premature vascular disease and controls. 13 vascular patients were selected from a previous study (Engbersen et al., Am. J. Hum. Genet., 1995, 56:142-150), among which 5 were considered to have thermolabile MTHFR. From a large reference group of 89 controls, all 7 individuals who had thermolabile MTHFR were studied, and an additional 20 controls with normal MTHFR were selected from the same reference group. Table 3 documents the relationship between genotypes and specific enzyme activity, thermolability and plasma homocysteine level. The mean MTHFR activity for individuals homozygous for the substitution (+/+) was approximately 30% of the mean activity for (-/-) individuals, homozygous for the alanine residue. Heterozygotes had a mean MTHFR activity that was 65% of the activity of (-/-) individuals; this value is intermediate between the values for (-/-) and (+/+) individuals. The ranges of activities showed some overlap for the heterozygous and (-/-) genotypes, but homozygous (+/+) individuals showed virtually no overlap with the former groups. A one-way analysis of variance yielded a p value <0.0001; a pairwise Bonferroni t test showed that all three genotypes were significantly different with p<0.01 for the three possible combinations.

The three genotypes were all significantly different (p<0.01) with respect to enzyme thermolability. The mean residual activity after heat inactivation for 5 minutes at 46° was 67%, 56% and 22% for the (-/-), (+/-) and (+/+) genotypes, respectively. While the degree of thermolability overlaps somewhat for (-/-) individuals and heterozygotes, individuals with two mutant alleles had a distinctly lower range. Every individual with the (+/+) genotype had residual activity <35% after heating, and specific activity <50% of that of the (-/-) genotype.

Total homocysteine concentrations, after fasting and 6 hours after methionine loading, were measured in plasma by high performance liquid chromatography using fluorescence detection. Fasting homocysteine levels in (+/+) individuals were almost twice the value for (+/-) and (-/-) individuals. The differences among genotypes for plasma homocysteine were maintained when homocysteine was measured following 6 hours of methionine loading. A one-way anova yielded a p<0.01 for the fasting and post-methionine homocysteine levels. A pairwise Bonferroni t test showed that only homozygous mutant individuals had significantly elevated homocysteine levels (p<0.05).

PCR-based Mutagenesis for Expression of A→V Mutation in vitro

PCR-based mutagenesis, using the cDNA-containing Bluescript™ vector as template, was used to create the A to V mutation. Vent™ polymerase (NEB) was used to reduce PCR errors. The following primers were used: primer 1, bp -200 to -178, sense; primer 2, bp 667 to 687, antisense, containing a mismatch, A, at bp 677; primer 3, 667 to 687, sense, containing a mismatch, T, at bp 677; primer 4, bp 1092 to 1114, antisense. PCR was performed using primers 1 and 2 to generate a product of 887 bp, and using primers 3 and 4 to generate a product of 447 bp. The two PCR fragments were isolated from a 1.2% agarose gel by Geneclean™ (BIO 101). A final PCR reaction, using primers 1 and 4 and the first 2 PCR fragments as template, was performed to generate a 1.3 kb band containing the mutation. The 1.3 kb fragment was digested with NcoI and MscI, and inserted into the wild-type cDNA- containing expression vector by replacing the sequences between the NcoI site at bp 11 and the MscI site at bp 943. The entire replacement fragment and the cloning sites were sequenced to verify that no additional changes were introduced by PCR.

Expression Analysis of Wild-Type and Mutagenized cDNA

Overnight cultures of JM105™ containing vector alone, vector+wild-type MTHFR cDNA, or vector+mutagenized cDNA were grown at 37° C. in 2×YT media with 0.05 mg/ml ampicillin. Fresh 10 ml. cultures of each were inoculated with approximately 50 μL of over-night cultures for a starting O.D. of 0.05, and were grown at 37° C. to an O.D. of 1 at 420 nM. Cultures were then induced for 2 hrs. with 1 mM IPTG and pelletted. The cells were resuspended in TE buffer with 2 μg/ml aprotinin and 2 μg/ml leupeptin (3.5×wet weight of cells). Cell suspensions were sonicated on ice for 3×15 sec. to break open cell membranes and then centrifuged for 30 mins. at 4° C. to pellet cell debris and unlysed cells. The supernatant was removed and assayed for protein concentration with the Bio-Rad™ protein assay. Western analysis was performed using the Amersham ECL™ kit according to the instructions of the supplier, using antiserum generated against purified porcine liver MTHFR. Enzymatic assays were performed by established procedures; thermolability was assessed by pre-treating the extracts at 46° C. for 5 mins. before determining activity. Specific activities (nmol formaldehyde/hr./mg. protein) were calculated for the 2 cDNA-containing constructs after subtraction of the values obtained with vector alone (to subtract background E. coli MTHFR activity).

The MTHFR cDNA (2.2 kb) (FIG. 6) has an open reading frame of 1980 bp, predicting a protein of 74.6 kDa. The purified porcine liver enzyme has been shown to have subunits of 77 kDa. Western analysis (FIG. 7A) of several human tissues and of porcine liver has revealed a polypeptide of 77 kDa in all the studied tissues, as well as an additional polypeptide of approximately 70 kDa in human fetal liver and in porcine liver, suggesting the presence of isozymes. Two μg of bacterial extract protein was used for lanes 1-3. The tissues (lanes 4-8) were prepared by homogenization in 0.25M sucrose with protease inhibitors (2 μg/ml each of aprotinin and leupeptin), followed by sonication (3×15 sec.) on ice. The extracts were spun for 15 min. in a microcentrifuge at 14,000 g and 100 μg of supernatant protein was used for Western analysis. h=human; p=porcine.

The wild-type cDNA and a mutagenized cDNA, containing the A→V substitution, were expressed in E. coli to yield a protein of approximately 70 kDa (FIG. 7A), which co-migrates with the smaller polypeptide mentioned above. Treatment of extracts at 46° C. for 5 minutes revealed that the enzyme containing the substitution was significantly more thermolabile than the wild-type enzyme (p<0.001; FIG. 7B). Two separate experiments (with 3-4 replicates for each construct for each experiment) were performed to measure thermostable activity of the wild-type MTHFR and mutagenized MTHFR A→V cDNAs. The values shown represent mean ±standard error for each experiment, as % of residual activity after heating. The means of the specific activities before heating (expressed as nmol formaldehyde/hr./mg. protein) were as follows: Exp. 1, 3.8 and 5.3 for MTHFR and MTHFR A→V, respectively; Exp. 2, 6.2 and 7.5 for MTHFR and MTHFR A→V, respectively. The expression experiments were not designed to measure differences in specific activity before heating, since variation in efficiencies of expression could contribute to difficulties in interpretation. Curiously though, the specific activity for the mutant construct was higher in both experiments. It is possible that the mutant protein has increased stability in E. coli, or that inclusion bodies in our extracts contributed to differences in recovery of properly-assembled enzyme.

These studies have identified a common substitution in the MTHFR gene which results in thermolability in vitro and in vivo. The mutation, in the heterozygous or homozygous state, correlates with reduced enzyme activity and increased thermolability of MTHFR in lymphocyte extracts. A significant elevation in plasma homocysteine was observed in individuals who were homozygous for the mutation. Statistically-significant differences for homocysteine levels were not observed between heterozygotes and (-/-) individuals; this observation is not surprising, since plasma homocysteine can be influenced by several environmental factors, including intake of folate, vitamin B₁₂, vitamin B₆, and methionine, as well as by genetic variation at other loci, such as the cystathionine-β-synthase gene.

The alanine to valine substitution conserves the hydrophobicity of the residue and is associated with small changes in activity, in contrast to non-conservative changes, such as the previously-reported arginine to glutamine change in MTHFR, which is associated with a greater decrease in enzyme activity and severe hyperhomocysteinemia. The alanine residue is situated in a region of homology with the bacterial metF genes. We have also observed the same region of homology in the human dihydrofolate reductase (DHFR) gene (FIG. 11), although the alanine residue itself is not conserved; this region of amino acids 130-149 of DHFR contains T136 which has been implicated in folate binding in an analysis of the crystal structure of recombinant human DHFR. It is tempting to speculate that this region in MTHFR is also involved in folate binding and that the enzyme may be stabilized in the presence of folate. This hypothesis is compatible with the well-documented influence of folate on homocysteine levels and with the reported correction of mild hyperhomocysteinemia by folic acid in individuals with premature vascular disease, and in individuals with thermolabile MTHFR.

Although our cDNA is not long enough to encode the larger MTHFR polypeptide, it is capable of directing synthesis of the smaller isozyme. The ATG start codon for this polypeptide is within a good consensus sequence for translation initiation. Whether the isozyme is restricted to liver and what its role is in this tissue remain to be determined.

These data have identified a common genetic change in MTHFR which results in thermolability; our experiments do not directly address the relationship between this change and vascular disease. Nonetheless, this polymorphism represents a diagnostic test for evaluation of MTHFR thermolability in hyperhomocysteinemia. Large case-control studies are required to evaluate the frequency of this genetic change in various forms of occlusive arterial disease and to examine the interaction between this genetic marker and dietary factors. Well-defined populations need to be examined, since the limited data set thus far suggests that population-specific allele frequencies may exist. More importantly, however, the identification of a candidate genetic risk factor for vascular disease, which may be influenced by nutrient intake, represents a critical step in the design of appropriate therapies for the homocysteinemic form of arteriosclerosis.

cDNA FOR MTHFR AND ITS POTENTIAL UTILITY

A human cDNA for MTHFR (2.2 kb) has been isolated, as reported by us in Goyette et al. (Nature Genetics, 1994, 7:195-200) and Frosst et al. (Nature Genetics, 1995, 10:111-113). The cDNA has been expressed in vitro to yield a MTHFR protein of approximately 70 kDa (Frosst P et al., Nature Genetics, 1995, 10:111-113).

Using the cDNA sequence, mutations in patients with severe and mild MTHFR deficiency (Goyette P et al., Nature Genetics, 1994, 7:195-200; Goyette P et al., Am. J. Hum. Genet., 1995, 56:1052-1059; Frosst P et al., Nature Genetics, 1995, 10:111-113) were identified.

The cDNA sequence is a necessary starting point for the detection of MTHFR sequence changes that would identify individuals at risk for cardiovascular and neurological diseases, as well as other disorders affected by folic acid metabolism. Diagnostic tests by DNA analysis are more efficient and accurate than testing by enzymatic/biochemical assays. Less blood is required and results are available in a shorter period of time. The tests could be performed as a routine operation in any laboratory that performs molecular genetic diagnosis, without the specialized reagents/expertise that is required for an enzymebased test.

The second major utility of the cDNA would be in the design of therapeutic protocols, for correction of MTHFR deficiency. These protocols could directly involve the gene, as in gene therapy trials or in the use of reagents that could modify gene expression. Alternatively, the therapy might require knowledge of the amino acid sequence (derived from the cDNA sequence), as in the use of reagents that would modify enzyme activity. The identification of sequences and/or sequence changes in specific regions of the cDNA or protein, such as FAD binding sites or folate-binding sites, are useful in designing therapeutic protocols involving the above nutrients.

UTILITY OF INVENTION IN CLINICAL AND DIAGNOSTIC STUDIES

Coronary artery disease patients in Montreal (n=153) were studied to examine the frequency of the alanine to valine substitution. Fourteen percent of the patients were homozygous for this mutation. An analysis of 70 control individuals (free of cardiovascular disease) demonstrated that only seven % of these individuals were homozygous for the alanine to valine mutation.

Analysis of homocysteine levels in 123 men of the above patient group indicated that the mutant allele significantly raised homocysteine levels from 10.2 micromoles/L in homozygous normal men to 11.5 and 12.7 in heterozygotes and homozygous mutants, respectively.

Families with a child with spina bifida, a neural tube defect, have been examined for the presence of the alanine to valine mutation. Approximately 16% of mothers who had a child with spina bifida were homozygous for this mutation, while only 5% of control individuals were homozygous. Fathers of children with spina bifida also had an increased prevalence of the homozygous mutant genotype (10%) as did the affected children themselves (13%).

Table 4 indicates the interactive effect of folic acid with the homozygous mutant alanine to valine change. In a study of families from Framingham, Mass. and Utah, individuals who were homozygous mutant but had folate levels above 5 ng/ml did not have increased homocysteine levels compared to individuals with the normal or heterozygous genotype. However, individuals who were homozygous mutant but had folate levels below 5 ng/ml had homocysteine levels that were significantly higher than the other genotypes.

                  TABLE 4                                                          ______________________________________                                         Mean fasting and PML homocysteine levels for different                           MTHFR genotypes                                                                         MTHFR genotype                                                      Plasma     Normals  Heterozygotes                                                                             Homozygotes                                       Homocysteine (-/-) (+/-) (+/+) P.sub.trend                                   ______________________________________                                         N          58       61         30                                                Fasting* 9.4 9.2 12.1 0.02                                                     Folate < 5 ng/mL 10.2 10.4 15.2  0.002                                         Folate ≧ 5 ng/mL 8.2 7.5 7.5 0.52                                       Post-Methionine 30.0 30.9 31.3 0.62                                            load                                                                         ______________________________________                                          *Significant interaction between folate levels and genotype (p = 0.03)   

Example III provides preliminary data for therapeutic intervention by folic acid supplementation to individuals who are homozygous for the alanine to valine change. The data suggest that higher levels of plasma folate would lead to normalization of homocysteine levels in mutant individuals and might prevent the occurrence of disorders associated with high homocysteine levels, such as cardiovascular disease, neural tube defects, and possibly other disorders. Folic acid supplementation for mutant individuals might also restore methionine and S-adenosylmethionine levels to normal. This would be relevant for disorders that are influenced by methylation, such as neoplasias, developmental anomalies, neurologic disease, etc.

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.

    __________________________________________________________________________     #             SEQUENCE LISTING                                                    - -  - - (1) GENERAL INFORMATION:                                              - -    (iii) NUMBER OF SEQUENCES: 14                                           - -  - - (2) INFORMATION FOR SEQ ID NO: 1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 2220 base - #pairs                                                 (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: DNA (genomic)                                      - -    (iii) HYPOTHETICAL: NO                                                  - -     (iv) ANTI-SENSE: NO                                                    - -     (ix) FEATURE:                                                                   (A) NAME/KEY: CDS                                                              (B) LOCATION:1..1980                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #1:                            - - AAT TCC GGA GCC ATG GTG AAC GAA GCC AGA GG - #A AAC AGC AGC CTC         AAC       48                                                                     Asn Ser Gly Ala Met Val Asn Glu Ala Arg Gl - #y Asn Ser Ser Leu Asn             1               5 - #                 10 - #                 15               - - CCC TGC TTG GAG GGC AGT GCC AGC AGT GGC AG - #T GAG AGC TCC AAA GAT            96                                                                        Pro Cys Leu Glu Gly Ser Ala Ser Ser Gly Se - #r Glu Ser Ser Lys Asp                         20     - #             25     - #             30                   - - AGT TCG AGA TGT TCC ACC CCG GGC CTG GAC CC - #T GAG CGG CAT GAG AGA           144                                                                        Ser Ser Arg Cys Ser Thr Pro Gly Leu Asp Pr - #o Glu Arg His Glu Arg                     35         - #         40         - #         45                       - - CTC CGG GAG AAG ATG AGG CGG CGA TTG GAA TC - #T GGT GAC AAG TGG TTC           192                                                                        Leu Arg Glu Lys Met Arg Arg Arg Leu Glu Se - #r Gly Asp Lys Trp Phe                 50             - #     55             - #     60                           - - TCC CTG GAA TTC TTC CCT CCT CGA ACT GCT GA - #G GGA GCT GTC AAT CTC           240                                                                        Ser Leu Glu Phe Phe Pro Pro Arg Thr Ala Gl - #u Gly Ala Val Asn Leu             65                 - # 70                 - # 75                 - # 80        - - ATC TCA AGG TTT GAC CGG ATG GCA GCA GGT GG - #C CCC CTC TAC ATA GAC           288                                                                        Ile Ser Arg Phe Asp Arg Met Ala Ala Gly Gl - #y Pro Leu Tyr Ile Asp                             85 - #                 90 - #                 95               - - GTG ACC TGG CAC CCA GCA GGT GAC CCT GGC TC - #A GAC AAG GAG ACC TCC           336                                                                        Val Thr Trp His Pro Ala Gly Asp Pro Gly Se - #r Asp Lys Glu Thr Ser                        100      - #           105      - #           110                   - - TCC ATG ATG ATC GCC AGC ACC GCC GTG AAC TA - #C TGT GGC CTG GAG ACC           384                                                                        Ser Met Met Ile Ala Ser Thr Ala Val Asn Ty - #r Cys Gly Leu Glu Thr                    115          - #       120          - #       125                       - - ATC CTG CAC ATG ACC TGC TGC CGT CAG CGC CT - #G GAG GAG ATC ACG GGC           432                                                                        Ile Leu His Met Thr Cys Cys Arg Gln Arg Le - #u Glu Glu Ile Thr Gly                130              - #   135              - #   140                           - - CAT CTG CAC AAA GCT AAG CAG CTG GGC CTG AA - #G AAC ATC ATG GCG CTG           480                                                                        His Leu His Lys Ala Lys Gln Leu Gly Leu Ly - #s Asn Ile Met Ala Leu            145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - CGG GGA GAC CCA ATA GGT GAC CAG TGG GAA GA - #G GAG GAG GGA GGC         TTC      528                                                                     Arg Gly Asp Pro Ile Gly Asp Gln Trp Glu Gl - #u Glu Glu Gly Gly Phe                           165  - #               170  - #               175               - - AAC TAC GCA GTG GAC CTG GTG AAG CAC ATC CG - #A AGT GAG TTT GGT GAC           576                                                                        Asn Tyr Ala Val Asp Leu Val Lys His Ile Ar - #g Ser Glu Phe Gly Asp                        180      - #           185      - #           190                   - - TAC TTT GAC ATC TGT GTG GCA GGT TAC CCC AA - #A GGC CAC CCC GAA GCA           624                                                                        Tyr Phe Asp Ile Cys Val Ala Gly Tyr Pro Ly - #s Gly His Pro Glu Ala                    195          - #       200          - #       205                       - - GGG AGC TTT GAG GCT GAC CTG AAG CAC TTG AA - #G GAG AAG GTG TCT GCG           672                                                                        Gly Ser Phe Glu Ala Asp Leu Lys His Leu Ly - #s Glu Lys Val Ser Ala                210              - #   215              - #   220                           - - GGA GCC GAT TTC ATC ATC ACG CAG CTT TTC TT - #T GAG GCT GAC ACA TTC           720                                                                        Gly Ala Asp Phe Ile Ile Thr Gln Leu Phe Ph - #e Glu Ala Asp Thr Phe            225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - TTC CGC TTT GTG AAG GCA TGC ACC GAC ATG GG - #C ATC ACT TGC CCC         ATC      768                                                                     Phe Arg Phe Val Lys Ala Cys Thr Asp Met Gl - #y Ile Thr Cys Pro Ile                           245  - #               250  - #               255               - - GTC CCC GGG ATC TTT CCC ATC CAG GGC TAC CA - #C TCC CTT CGG CAG CTT           816                                                                        Val Pro Gly Ile Phe Pro Ile Gln Gly Tyr Hi - #s Ser Leu Arg Gln Leu                        260      - #           265      - #           270                   - - GTG AAG CTG TCC AAG CTG GAG GTG CCA CAG GA - #G ATC AAG GAC GTG ATT           864                                                                        Val Lys Leu Ser Lys Leu Glu Val Pro Gln Gl - #u Ile Lys Asp Val Ile                    275          - #       280          - #       285                       - - GAG CCA ATC AAA GAC AAC GAT GCT GCC ATC CG - #C AAC TAT GGC ATC GAG           912                                                                        Glu Pro Ile Lys Asp Asn Asp Ala Ala Ile Ar - #g Asn Tyr Gly Ile Glu                290              - #   295              - #   300                           - - CTG GCC GTG AGC CTG TGC CAG GAG CTT CTG GC - #C AGT GGC TTG GTG CCA           960                                                                        Leu Ala Val Ser Leu Cys Gln Glu Leu Leu Al - #a Ser Gly Leu Val Pro            305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - GGC CTC CAC TTC TAC ACC CTC AAC CGC GAG AT - #G GCT ACC ACA GAG         GTG     1008                                                                     Gly Leu His Phe Tyr Thr Leu Asn Arg Glu Me - #t Ala Thr Thr Glu Val                           325  - #               330  - #               335               - - CTG AAG CGC CTG GGG ATG TGG ACT GAG GAC CC - #C AGG CGT CCC CTA CCC          1056                                                                        Leu Lys Arg Leu Gly Met Trp Thr Glu Asp Pr - #o Arg Arg Pro Leu Pro                        340      - #           345      - #           350                   - - TGG GCT CTC AGT GCC CAC CCC AAG CGC CGA GA - #G GAA GAT GTA CGT CCC          1104                                                                        Trp Ala Leu Ser Ala His Pro Lys Arg Arg Gl - #u Glu Asp Val Arg Pro                    355          - #       360          - #       365                       - - ATC TTC TGG GCC TCC AGA CCA AAG AGT TAC AT - #C TAC CGT ACC CAG GAG          1152                                                                        Ile Phe Trp Ala Ser Arg Pro Lys Ser Tyr Il - #e Tyr Arg Thr Gln Glu                370              - #   375              - #   380                           - - TGG GAC GAG TTC CCT AAC GGC CGC TGG GGC AA - #T TCC TCT TCC CCT GCC          1200                                                                        Trp Asp Glu Phe Pro Asn Gly Arg Trp Gly As - #n Ser Ser Ser Pro Ala            385                 3 - #90                 3 - #95                 4 -       #00                                                                               - - TTT GGG GAG CTG AAG GAC TAC TAC CTC TTC TA - #C CTG AAG AGC AAG         TCC     1248                                                                     Phe Gly Glu Leu Lys Asp Tyr Tyr Leu Phe Ty - #r Leu Lys Ser Lys Ser                           405  - #               410  - #               415               - - CCC AAG GAG GAG CTG CTG AAG ATG TGG GGG GA - #G GAG CTG ACC AGT GAA          1296                                                                        Pro Lys Glu Glu Leu Leu Lys Met Trp Gly Gl - #u Glu Leu Thr Ser Glu                        420      - #           425      - #           430                   - - GCA AGT GTC TTT GAA GTC TTT GTT CTT TAC CT - #C TCG GGA GAA CCA AAC          1344                                                                        Ala Ser Val Phe Glu Val Phe Val Leu Tyr Le - #u Ser Gly Glu Pro Asn                    435          - #       440          - #       445                       - - CGG AAT GGT CAC AAA GTG ACT TGC CTG CCC TG - #G AAC GAT GAG CCC CTG          1392                                                                        Arg Asn Gly His Lys Val Thr Cys Leu Pro Tr - #p Asn Asp Glu Pro Leu                450              - #   455              - #   460                           - - GCG GCT GAG ACC AGC CTG CTG AAG GAG GAG CT - #G CTG CGG GTG AAC CGC          1440                                                                        Ala Ala Glu Thr Ser Leu Leu Lys Glu Glu Le - #u Leu Arg Val Asn Arg            465                 4 - #70                 4 - #75                 4 -       #80                                                                               - - CAG GGC ATC CTC ACC ATC AAC TCA CAG CCC AA - #C ATC AAC GGG AAG         CCG     1488                                                                     Gln Gly Ile Leu Thr Ile Asn Ser Gln Pro As - #n Ile Asn Gly Lys Pro                           485  - #               490  - #               495               - - TCC TCC GAC CCC ATC GTG GGC TGG GGC CCC AG - #C GGG GGC TAT GTC TTC          1536                                                                        Ser Ser Asp Pro Ile Val Gly Trp Gly Pro Se - #r Gly Gly Tyr Val Phe                        500      - #           505      - #           510                   - - CAG AAG GCC TAC TTA GAG TTT TTC ACT TCC CG - #C GAG ACA GCG GAA GCA          1584                                                                        Gln Lys Ala Tyr Leu Glu Phe Phe Thr Ser Ar - #g Glu Thr Ala Glu Ala                    515          - #       520          - #       525                       - - CTT CTG CAA GTG CTG AAG AAG TAC GAG CTC CG - #G GTT AAT TAC CAC CTT          1632                                                                        Leu Leu Gln Val Leu Lys Lys Tyr Glu Leu Ar - #g Val Asn Tyr His Leu                530              - #   535              - #   540                           - - GTC AAT GTG AAG GGT GAA AAC ATC ACC AAT GC - #C CCT GAA CTG CAG CCG          1680                                                                        Val Asn Val Lys Gly Glu Asn Ile Thr Asn Al - #a Pro Glu Leu Gln Pro            545                 5 - #50                 5 - #55                 5 -       #60                                                                               - - AAT GCT GTC ACT TGG GGC ATC TTC CCT GGG CG - #A GAG ATC ATC CAG         CCC     1728                                                                     Asn Ala Val Thr Trp Gly Ile Phe Pro Gly Ar - #g Glu Ile Ile Gln Pro                           565  - #               570  - #               575               - - ACC GTA GTG GAT CCC GTC AGC TTC ATG TTC TG - #G AAG GAC GAG GCC TTT          1776                                                                        Thr Val Val Asp Pro Val Ser Phe Met Phe Tr - #p Lys Asp Glu Ala Phe                        580      - #           585      - #           590                   - - GCC CTG TGG ATT GAG CGG TGG GGA AAG CTG TA - #T GAG GAG GAG TCC CCG          1824                                                                        Ala Leu Trp Ile Glu Arg Trp Gly Lys Leu Ty - #r Glu Glu Glu Ser Pro                    595          - #       600          - #       605                       - - TCC CGC ACC ATC ATC CAG TAC ATC CAC GAC AA - #C TAC TTC CTG GTC AAC          1872                                                                        Ser Arg Thr Ile Ile Gln Tyr Ile His Asp As - #n Tyr Phe Leu Val Asn                610              - #   615              - #   620                           - - CTG GTG GAC AAT GAC TTC CCA CTG GAC AAC TG - #C CTC TGG CAG GTG GTG          1920                                                                        Leu Val Asp Asn Asp Phe Pro Leu Asp Asn Cy - #s Leu Trp Gln Val Val            625                 6 - #30                 6 - #35                 6 -       #40                                                                               - - GAA GAC ACA TTG GAG CTT CTC AAC AGG CCC AC - #C CAG AAT GCG AGA         GAA     1968                                                                     Glu Asp Thr Leu Glu Leu Leu Asn Arg Pro Th - #r Gln Asn Ala Arg Glu                           645  - #               650  - #               655               - - ACG GAG GCT CCA TGACCCTGCG TCCTGACGCC CTGCGTTGGA GC - #CACTCCTG              2020                                                                        Thr Glu Ala Pro                                                                            660                                                                 - - TCCCGCCTTC CTCCTCCACA GTGCTGCTTC TCTTGGGAAC TCCACTCTCC TT -              #CGTGTCTC   2080                                                                  - - TCCCACCCCG GCCTCCACTC CCCCACCTGA CAATGGCAGC TAGACTGGAG TG -             #AGGCTTCC   2140                                                                  - - AGGCTCTTCC TGGACCTGAG TCGGCCCCAC ATGGGAACCT AGTACTCTCT GC -             #TCTAAAAA   2200                                                                  - - AAAAAAAAAA AAAGGAATTC            - #                  - #                      222 - #0                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO: 2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 660 amino - #acids                                                 (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #2:                            - - Asn Ser Gly Ala Met Val Asn Glu Ala Arg Gl - #y Asn Ser Ser Leu Asn         1               5 - #                 10 - #                 15               - - Pro Cys Leu Glu Gly Ser Ala Ser Ser Gly Se - #r Glu Ser Ser Lys Asp                    20     - #             25     - #             30                   - - Ser Ser Arg Cys Ser Thr Pro Gly Leu Asp Pr - #o Glu Arg His Glu Arg                35         - #         40         - #         45                       - - Leu Arg Glu Lys Met Arg Arg Arg Leu Glu Se - #r Gly Asp Lys Trp Phe            50             - #     55             - #     60                           - - Ser Leu Glu Phe Phe Pro Pro Arg Thr Ala Gl - #u Gly Ala Val Asn Leu        65                 - # 70                 - # 75                 - # 80        - - Ile Ser Arg Phe Asp Arg Met Ala Ala Gly Gl - #y Pro Leu Tyr Ile Asp                        85 - #                 90 - #                 95               - - Val Thr Trp His Pro Ala Gly Asp Pro Gly Se - #r Asp Lys Glu Thr Ser                   100      - #           105      - #           110                   - - Ser Met Met Ile Ala Ser Thr Ala Val Asn Ty - #r Cys Gly Leu Glu Thr               115          - #       120          - #       125                       - - Ile Leu His Met Thr Cys Cys Arg Gln Arg Le - #u Glu Glu Ile Thr Gly           130              - #   135              - #   140                           - - His Leu His Lys Ala Lys Gln Leu Gly Leu Ly - #s Asn Ile Met Ala Leu       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Arg Gly Asp Pro Ile Gly Asp Gln Trp Glu Gl - #u Glu Glu Gly Gly         Phe                                                                                              165  - #               170  - #               175              - - Asn Tyr Ala Val Asp Leu Val Lys His Ile Ar - #g Ser Glu Phe Gly Asp                   180      - #           185      - #           190                   - - Tyr Phe Asp Ile Cys Val Ala Gly Tyr Pro Ly - #s Gly His Pro Glu Ala               195          - #       200          - #       205                       - - Gly Ser Phe Glu Ala Asp Leu Lys His Leu Ly - #s Glu Lys Val Ser Ala           210              - #   215              - #   220                           - - Gly Ala Asp Phe Ile Ile Thr Gln Leu Phe Ph - #e Glu Ala Asp Thr Phe       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Phe Arg Phe Val Lys Ala Cys Thr Asp Met Gl - #y Ile Thr Cys Pro         Ile                                                                                              245  - #               250  - #               255              - - Val Pro Gly Ile Phe Pro Ile Gln Gly Tyr Hi - #s Ser Leu Arg Gln Leu                   260      - #           265      - #           270                   - - Val Lys Leu Ser Lys Leu Glu Val Pro Gln Gl - #u Ile Lys Asp Val Ile               275          - #       280          - #       285                       - - Glu Pro Ile Lys Asp Asn Asp Ala Ala Ile Ar - #g Asn Tyr Gly Ile Glu           290              - #   295              - #   300                           - - Leu Ala Val Ser Leu Cys Gln Glu Leu Leu Al - #a Ser Gly Leu Val Pro       305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - Gly Leu His Phe Tyr Thr Leu Asn Arg Glu Me - #t Ala Thr Thr Glu         Val                                                                                              325  - #               330  - #               335              - - Leu Lys Arg Leu Gly Met Trp Thr Glu Asp Pr - #o Arg Arg Pro Leu Pro                   340      - #           345      - #           350                   - - Trp Ala Leu Ser Ala His Pro Lys Arg Arg Gl - #u Glu Asp Val Arg Pro               355          - #       360          - #       365                       - - Ile Phe Trp Ala Ser Arg Pro Lys Ser Tyr Il - #e Tyr Arg Thr Gln Glu           370              - #   375              - #   380                           - - Trp Asp Glu Phe Pro Asn Gly Arg Trp Gly As - #n Ser Ser Ser Pro Ala       385                 3 - #90                 3 - #95                 4 -       #00                                                                               - - Phe Gly Glu Leu Lys Asp Tyr Tyr Leu Phe Ty - #r Leu Lys Ser Lys         Ser                                                                                              405  - #               410  - #               415              - - Pro Lys Glu Glu Leu Leu Lys Met Trp Gly Gl - #u Glu Leu Thr Ser Glu                   420      - #           425      - #           430                   - - Ala Ser Val Phe Glu Val Phe Val Leu Tyr Le - #u Ser Gly Glu Pro Asn               435          - #       440          - #       445                       - - Arg Asn Gly His Lys Val Thr Cys Leu Pro Tr - #p Asn Asp Glu Pro Leu           450              - #   455              - #   460                           - - Ala Ala Glu Thr Ser Leu Leu Lys Glu Glu Le - #u Leu Arg Val Asn Arg       465                 4 - #70                 4 - #75                 4 -       #80                                                                               - - Gln Gly Ile Leu Thr Ile Asn Ser Gln Pro As - #n Ile Asn Gly Lys         Pro                                                                                              485  - #               490  - #               495              - - Ser Ser Asp Pro Ile Val Gly Trp Gly Pro Se - #r Gly Gly Tyr Val Phe                   500      - #           505      - #           510                   - - Gln Lys Ala Tyr Leu Glu Phe Phe Thr Ser Ar - #g Glu Thr Ala Glu Ala               515          - #       520          - #       525                       - - Leu Leu Gln Val Leu Lys Lys Tyr Glu Leu Ar - #g Val Asn Tyr His Leu           530              - #   535              - #   540                           - - Val Asn Val Lys Gly Glu Asn Ile Thr Asn Al - #a Pro Glu Leu Gln Pro       545                 5 - #50                 5 - #55                 5 -       #60                                                                               - - Asn Ala Val Thr Trp Gly Ile Phe Pro Gly Ar - #g Glu Ile Ile Gln         Pro                                                                                              565  - #               570  - #               575              - - Thr Val Val Asp Pro Val Ser Phe Met Phe Tr - #p Lys Asp Glu Ala Phe                   580      - #           585      - #           590                   - - Ala Leu Trp Ile Glu Arg Trp Gly Lys Leu Ty - #r Glu Glu Glu Ser Pro               595          - #       600          - #       605                       - - Ser Arg Thr Ile Ile Gln Tyr Ile His Asp As - #n Tyr Phe Leu Val Asn           610              - #   615              - #   620                           - - Leu Val Asp Asn Asp Phe Pro Leu Asp Asn Cy - #s Leu Trp Gln Val Val       625                 6 - #30                 6 - #35                 6 -       #40                                                                               - - Glu Asp Thr Leu Glu Leu Leu Asn Arg Pro Th - #r Gln Asn Ala Arg         Glu                                                                                              645  - #               650  - #               655              - - Thr Glu Ala Pro                                                                       660                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO: 3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 2219 base - #pairs                                                 (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: DNA (genomic)                                      - -    (iii) HYPOTHETICAL: NO                                                  - -     (iv) ANTI-SENSE: NO                                                    - -     (ix) FEATURE:                                                                   (A) NAME/KEY: CDS                                                              (B) LOCATION:13..1983                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #3:                            - - AATTCCGGAG CC ATG GTG AAC GAA GCC AGA GGA AAC - # AGC AGC CTC AAC              48                                                                                      Met Val As - #n Glu Ala Arg Gly Asn Ser Ser Leu Asn                                - #            665     - #            670                     - - CCC TGC TTG GAG GGC AGT GCC AGC AGT GGC AG - #T GAG AGC TCC AAA GAT            96                                                                        Pro Cys Leu Glu Gly Ser Ala Ser Ser Gly Se - #r Glu Ser Ser Lys Asp                    675          - #       680          - #       685                       - - AGT TCG AGA TGT TCC ACC CCG GGC CTG GAC CC - #T GAG CGG CAT GAG AGA           144                                                                        Ser Ser Arg Cys Ser Thr Pro Gly Leu Asp Pr - #o Glu Arg His Glu Arg                690              - #   695              - #   700                           - - CTC CGG GAG AAG ATG AGG CGG CGA TTG GAA TC - #T GGT GAC AAG TGG TTC           192                                                                        Leu Arg Glu Lys Met Arg Arg Arg Leu Glu Se - #r Gly Asp Lys Trp Phe            705                 7 - #10                 7 - #15                 7 -       #20                                                                               - - TCC CTG GAA TTC TTC CCT CCT CGA ACT GCT GA - #G GGA GCT GTC AAT         CTC      240                                                                     Ser Leu Glu Phe Phe Pro Pro Arg Thr Ala Gl - #u Gly Ala Val Asn Leu                           725  - #               730  - #               735               - - ATC TCA AGG TTT GAC CGG ATG GCA GCA GGT GG - #C CCC CTC TAC ATA GAC           288                                                                        Ile Ser Arg Phe Asp Arg Met Ala Ala Gly Gl - #y Pro Leu Tyr Ile Asp                        740      - #           745      - #           750                   - - GTG ACC TGG CAC CCA GCA GGT GAC CCT GGC TC - #A GAC AAG GAG ACC TCC           336                                                                        Val Thr Trp His Pro Ala Gly Asp Pro Gly Se - #r Asp Lys Glu Thr Ser                    755          - #       760          - #       765                       - - TCC ATG ATG ATC GCC AGC ACC GCC GTG AAC TA - #C TGT GGC CTG GAG ACC           384                                                                        Ser Met Met Ile Ala Ser Thr Ala Val Asn Ty - #r Cys Gly Leu Glu Thr                770              - #   775              - #   780                           - - ATC CTG CAC ATG ACC TGC TGC CGT CAG CGC CT - #G GAG GAG ATC ACG GGC           432                                                                        Ile Leu His Met Thr Cys Cys Arg Gln Arg Le - #u Glu Glu Ile Thr Gly            785                 7 - #90                 7 - #95                 8 -       #00                                                                               - - CAT CTG CAC AAA GCT AAG CAG CTG GGC CTG AA - #G AAC ATC ATG GCG         CTG      480                                                                     His Leu His Lys Ala Lys Gln Leu Gly Leu Ly - #s Asn Ile Met Ala Leu                           805  - #               810  - #               815               - - CGG GGA GAC CCA ATA GGT GAC CAG TGG GAA GA - #G GAG GAG GGA GGC TTC           528                                                                        Arg Gly Asp Pro Ile Gly Asp Gln Trp Glu Gl - #u Glu Glu Gly Gly Phe                        820      - #           825      - #           830                   - - AAC TAC GCA GTG GAC CTG GTG AAG CAC ATC CG - #A AGT GAG TTT GGT GAC           576                                                                        Asn Tyr Ala Val Asp Leu Val Lys His Ile Ar - #g Ser Glu Phe Gly Asp                    835          - #       840          - #       845                       - - TAC TTT GAC ATC TGT GTG GCA GGT TAC CCC AA - #A GGC CAC CCC GAA GCA           624                                                                        Tyr Phe Asp Ile Cys Val Ala Gly Tyr Pro Ly - #s Gly His Pro Glu Ala                850              - #   855              - #   860                           - - GGG AGC TTT GAG GCT GAC CTG AAG CAC TTG AA - #G GAG AAG GTG TCT GCG           672                                                                        Gly Ser Phe Glu Ala Asp Leu Lys His Leu Ly - #s Glu Lys Val Ser Ala            865                 8 - #70                 8 - #75                 8 -       #80                                                                               - - GGA GCC GAT TTC ATC ATC ACG CAG CTT TTC TT - #T GAG GCT GAC ACA         TTC      720                                                                     Gly Ala Asp Phe Ile Ile Thr Gln Leu Phe Ph - #e Glu Ala Asp Thr Phe                           885  - #               890  - #               895               - - TTC CGC TTT GTG AAG GCA TGC ACC GAC ATG GG - #C ATC ACT TGC CCC ATC           768                                                                        Phe Arg Phe Val Lys Ala Cys Thr Asp Met Gl - #y Ile Thr Cys Pro Ile                        900      - #           905      - #           910                   - - GTC CCC GGG ATC TTT CCC ATC CAG GGC TAC CA - #C TCC CTT CGG CAG CTT           816                                                                        Val Pro Gly Ile Phe Pro Ile Gln Gly Tyr Hi - #s Ser Leu Arg Gln Leu                    915          - #       920          - #       925                       - - GTG AAG CTG TCC AAG CTG GAG GTG CCA CAG GA - #G ATC AAG GAC GTG ATT           864                                                                        Val Lys Leu Ser Lys Leu Glu Val Pro Gln Gl - #u Ile Lys Asp Val Ile                930              - #   935              - #   940                           - - GAG CCA ATC AAA GAC AAC GAT GCT GCC ATC CG - #C AAC TAT GGC ATC GAG           912                                                                        Glu Pro Ile Lys Asp Asn Asp Ala Ala Ile Ar - #g Asn Tyr Gly Ile Glu            945                 9 - #50                 9 - #55                 9 -       #60                                                                               - - CTG GCC GTG AGC CTG TGC CAG GAG CTT CTG GC - #C AGT GGC TTG GTG         CCA      960                                                                     Leu Ala Val Ser Leu Cys Gln Glu Leu Leu Al - #a Ser Gly Leu Val Pro                           965  - #               970  - #               975               - - GGC CTC CAC TTC TAC ACC CTC AAC CGC GAG AT - #G GCT ACC ACA GAG GTG          1008                                                                        Gly Leu His Phe Tyr Thr Leu Asn Arg Glu Me - #t Ala Thr Thr Glu Val                        980      - #           985      - #           990                   - - CTG AAG CGC CTG GGG ATG TGG ACT GAG GAC CC - #C AGG CGT CCC CTA CCC          1056                                                                        Leu Lys Arg Leu Gly Met Trp Thr Glu Asp Pr - #o Arg Arg Pro Leu Pro                    995          - #       1000          - #      1005                      - - TGG GCT CTC AGT GCC CAC CCC AAG CGC CGA GA - #G GAA GAT GTA CGT CCC          1104                                                                        Trp Ala Leu Ser Ala His Pro Lys Arg Arg Gl - #u Glu Asp Val Arg Pro                1010             - #   1015              - #  1020                          - - ATC TTC TGG GCC TCC AGA CCA AAG AGT TAC AT - #C TAC CGT ACC CAG GAG          1152                                                                        Ile Phe Trp Ala Ser Arg Pro Lys Ser Tyr Il - #e Tyr Arg Thr Gln Glu            1025                1030 - #                1035 - #               1040         - - TGG GAC GAG TTC CCT AAC GGC CGC TGG GGC AA - #T TCC TCT TCC CCT GCC          1200                                                                        Trp Asp Glu Phe Pro Asn Gly Arg Trp Gly As - #n Ser Ser Ser Pro Ala                            1045 - #               1050  - #              1055              - - TTT GGG GAG CTG AAG GAC TAC TAC CTC TTC TA - #C CTG AAG AGC AAG TCC          1248                                                                        Phe Gly Glu Leu Lys Asp Tyr Tyr Leu Phe Ty - #r Leu Lys Ser Lys Ser                        1060     - #           1065      - #          1070                  - - CCC AAG GAG GAG CTG CTG AAG ATG TGG GGG GA - #G GAG CTG ACC AGT GAA          1296                                                                        Pro Lys Glu Glu Leu Leu Lys Met Trp Gly Gl - #u Glu Leu Thr Ser Glu                    1075         - #       1080          - #      1085                      - - GCA AGT GTC TTT GAA GTC TTT GTT CTT TAC CT - #C TCG GGA GAA CCA AAC          1344                                                                        Ala Ser Val Phe Glu Val Phe Val Leu Tyr Le - #u Ser Gly Glu Pro Asn                1090             - #   1095              - #  1100                          - - CGG AAT GGT CAC AAA GTG ACT TGC CTG CCC TG - #G AAC GAT GAG CCC CTG          1392                                                                        Arg Asn Gly His Lys Val Thr Cys Leu Pro Tr - #p Asn Asp Glu Pro Leu            1105                1110 - #                1115 - #               1120         - - GCG GCT GAG ACC AGC CTG CTG AAG GAG GAG CT - #G CTG CGG GTG AAC CGC          1440                                                                        Ala Ala Glu Thr Ser Leu Leu Lys Glu Glu Le - #u Leu Arg Val Asn Arg                            1125 - #               1130  - #              1135              - - CAG GGC ATC CTC ACC ATC AAC TCA CAG CCC AA - #C ATC AAC GGG AAG CCG          1488                                                                        Gln Gly Ile Leu Thr Ile Asn Ser Gln Pro As - #n Ile Asn Gly Lys Pro                        1140     - #           1145      - #          1150                  - - TCC TCC GAC CCC ATC GTG GGC TGG GGC CCC AG - #C GGG GGC TAT GTC TTC          1536                                                                        Ser Ser Asp Pro Ile Val Gly Trp Gly Pro Se - #r Gly Gly Tyr Val Phe                    1155         - #       1160          - #      1165                      - - CAG AAG GCC TAC TTA GAG TTT TTC ACT TCC CG - #C GAG ACA GCG GAA GCA          1584                                                                        Gln Lys Ala Tyr Leu Glu Phe Phe Thr Ser Ar - #g Glu Thr Ala Glu Ala                1170             - #   1175              - #  1180                          - - CTT CTG CAA GTG CTG AAG AAG TAC GAG CTC CG - #G GTT AAT TAC CAC CTT          1632                                                                        Leu Leu Gln Val Leu Lys Lys Tyr Glu Leu Ar - #g Val Asn Tyr His Leu            1185                1190 - #                1195 - #               1200         - - GTC AAT GTG AAG GGT GAA AAC ATC ACC AAT GC - #C CCT GAA CTG CAG CCG          1680                                                                        Val Asn Val Lys Gly Glu Asn Ile Thr Asn Al - #a Pro Glu Leu Gln Pro                            1205 - #               1210  - #              1215              - - AAT GCT GTC ACT TGG GGC ATC TTC CCT GGG CG - #A GAG ATC ATC CAG CCC          1728                                                                        Asn Ala Val Thr Trp Gly Ile Phe Pro Gly Ar - #g Glu Ile Ile Gln Pro                        1220     - #           1225      - #          1230                  - - ACC GTA GTG GAT CCC GTC AGC TTC ATG TTC TG - #G AAG GAC GAG GCC TTT          1776                                                                        Thr Val Val Asp Pro Val Ser Phe Met Phe Tr - #p Lys Asp Glu Ala Phe                    1235         - #       1240          - #      1245                      - - GCC CTG TGG ATT GAG CGG TGG GGA AAG CTG TA - #T GAG GAG GAG TCC CCG          1824                                                                        Ala Leu Trp Ile Glu Arg Trp Gly Lys Leu Ty - #r Glu Glu Glu Ser Pro                1250             - #   1255              - #  1260                          - - TCC CGC ACC ATC ATC CAG TAC ATC CAC GAC AA - #C TAC TTC CTG GTC AAC          1872                                                                        Ser Arg Thr Ile Ile Gln Tyr Ile His Asp As - #n Tyr Phe Leu Val Asn            1265                1270 - #                1275 - #               1280         - - CTG GTG GAC AAT GAC TTC CCA CTG GAC AAC TG - #C CTC TGG CAG GTG GTG          1920                                                                        Leu Val Asp Asn Asp Phe Pro Leu Asp Asn Cy - #s Leu Trp Gln Val Val                            1285 - #               1290  - #              1295              - - GAA GAC ACA TTG GAG CTT CTC AAC AGG CCC AC - #C CAG AAT GCG AGA GAA          1968                                                                        Glu Asp Thr Leu Glu Leu Leu Asn Arg Pro Th - #r Gln Asn Ala Arg Glu                        1300     - #           1305      - #          1310                  - - ACG GAG GCT CCA TGA CCCTGCGTCC TGACGCCCTG CGTTGGAGC - #C ACTCCTGTCC          2023                                                                        Thr Glu Ala Pro  *                                                                     1315                                                                    - - CGCCTTCCTC CTCCACAGTG CTGCTTCTCT TGGGAACTCC ACTCTCCTTC GT -              #GTCTCTCC   2083                                                                  - - CACCCCGGCC TCCACTCCCC CACCTGACAA TGGCAGCTAG ACTGGAGTGA GG -             #CTTCCAGG   2143                                                                  - - CTCTTCCTGG ACCTGAGTCG GCCCCACATG GGAACCTAGT ACTCTCTGCT CT -             #AAAAAAAA   2203                                                                  - - AAAAAAAAAA GGAATT             - #                  - #                       - #  2219                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 656 amino - #acids                                                 (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #4:                            - - Met Val Asn Glu Ala Arg Gly Asn Ser Ser Le - #u Asn Pro Cys Leu Glu         1               5 - #                 10 - #                 15               - - Gly Ser Ala Ser Ser Gly Ser Glu Ser Ser Ly - #s Asp Ser Ser Arg Cys                    20     - #             25     - #             30                   - - Ser Thr Pro Gly Leu Asp Pro Glu Arg His Gl - #u Arg Leu Arg Glu Lys                35         - #         40         - #         45                       - - Met Arg Arg Arg Leu Glu Ser Gly Asp Lys Tr - #p Phe Ser Leu Glu Phe            50             - #     55             - #     60                           - - Phe Pro Pro Arg Thr Ala Glu Gly Ala Val As - #n Leu Ile Ser Arg Phe        65                 - # 70                 - # 75                 - # 80        - - Asp Arg Met Ala Ala Gly Gly Pro Leu Tyr Il - #e Asp Val Thr Trp His                        85 - #                 90 - #                 95               - - Pro Ala Gly Asp Pro Gly Ser Asp Lys Glu Th - #r Ser Ser Met Met Ile                   100      - #           105      - #           110                   - - Ala Ser Thr Ala Val Asn Tyr Cys Gly Leu Gl - #u Thr Ile Leu His Met               115          - #       120          - #       125                       - - Thr Cys Cys Arg Gln Arg Leu Glu Glu Ile Th - #r Gly His Leu His Lys           130              - #   135              - #   140                           - - Ala Lys Gln Leu Gly Leu Lys Asn Ile Met Al - #a Leu Arg Gly Asp Pro       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Ile Gly Asp Gln Trp Glu Glu Glu Glu Gly Gl - #y Phe Asn Tyr Ala         Val                                                                                              165  - #               170  - #               175              - - Asp Leu Val Lys His Ile Arg Ser Glu Phe Gl - #y Asp Tyr Phe Asp Ile                   180      - #           185      - #           190                   - - Cys Val Ala Gly Tyr Pro Lys Gly His Pro Gl - #u Ala Gly Ser Phe Glu               195          - #       200          - #       205                       - - Ala Asp Leu Lys His Leu Lys Glu Lys Val Se - #r Ala Gly Ala Asp Phe           210              - #   215              - #   220                           - - Ile Ile Thr Gln Leu Phe Phe Glu Ala Asp Th - #r Phe Phe Arg Phe Val       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Lys Ala Cys Thr Asp Met Gly Ile Thr Cys Pr - #o Ile Val Pro Gly         Ile                                                                                              245  - #               250  - #               255              - - Phe Pro Ile Gln Gly Tyr His Ser Leu Arg Gl - #n Leu Val Lys Leu Ser                   260      - #           265      - #           270                   - - Lys Leu Glu Val Pro Gln Glu Ile Lys Asp Va - #l Ile Glu Pro Ile Lys               275          - #       280          - #       285                       - - Asp Asn Asp Ala Ala Ile Arg Asn Tyr Gly Il - #e Glu Leu Ala Val Ser           290              - #   295              - #   300                           - - Leu Cys Gln Glu Leu Leu Ala Ser Gly Leu Va - #l Pro Gly Leu His Phe       305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - Tyr Thr Leu Asn Arg Glu Met Ala Thr Thr Gl - #u Val Leu Lys Arg         Leu                                                                                              325  - #               330  - #               335              - - Gly Met Trp Thr Glu Asp Pro Arg Arg Pro Le - #u Pro Trp Ala Leu Ser                   340      - #           345      - #           350                   - - Ala His Pro Lys Arg Arg Glu Glu Asp Val Ar - #g Pro Ile Phe Trp Ala               355          - #       360          - #       365                       - - Ser Arg Pro Lys Ser Tyr Ile Tyr Arg Thr Gl - #n Glu Trp Asp Glu Phe           370              - #   375              - #   380                           - - Pro Asn Gly Arg Trp Gly Asn Ser Ser Ser Pr - #o Ala Phe Gly Glu Leu       385                 3 - #90                 3 - #95                 4 -       #00                                                                               - - Lys Asp Tyr Tyr Leu Phe Tyr Leu Lys Ser Ly - #s Ser Pro Lys Glu         Glu                                                                                              405  - #               410  - #               415              - - Leu Leu Lys Met Trp Gly Glu Glu Leu Thr Se - #r Glu Ala Ser Val Phe                   420      - #           425      - #           430                   - - Glu Val Phe Val Leu Tyr Leu Ser Gly Glu Pr - #o Asn Arg Asn Gly His               435          - #       440          - #       445                       - - Lys Val Thr Cys Leu Pro Trp Asn Asp Glu Pr - #o Leu Ala Ala Glu Thr           450              - #   455              - #   460                           - - Ser Leu Leu Lys Glu Glu Leu Leu Arg Val As - #n Arg Gln Gly Ile Leu       465                 4 - #70                 4 - #75                 4 -       #80                                                                               - - Thr Ile Asn Ser Gln Pro Asn Ile Asn Gly Ly - #s Pro Ser Ser Asp         Pro                                                                                              485  - #               490  - #               495              - - Ile Val Gly Trp Gly Pro Ser Gly Gly Tyr Va - #l Phe Gln Lys Ala Tyr                   500      - #           505      - #           510                   - - Leu Glu Phe Phe Thr Ser Arg Glu Thr Ala Gl - #u Ala Leu Leu Gln Val               515          - #       520          - #       525                       - - Leu Lys Lys Tyr Glu Leu Arg Val Asn Tyr Hi - #s Leu Val Asn Val Lys           530              - #   535              - #   540                           - - Gly Glu Asn Ile Thr Asn Ala Pro Glu Leu Gl - #n Pro Asn Ala Val Thr       545                 5 - #50                 5 - #55                 5 -       #60                                                                               - - Trp Gly Ile Phe Pro Gly Arg Glu Ile Ile Gl - #n Pro Thr Val Val         Asp                                                                                              565  - #               570  - #               575              - - Pro Val Ser Phe Met Phe Trp Lys Asp Glu Al - #a Phe Ala Leu Trp Ile                   580      - #           585      - #           590                   - - Glu Arg Trp Gly Lys Leu Tyr Glu Glu Glu Se - #r Pro Ser Arg Thr Ile               595          - #       600          - #       605                       - - Ile Gln Tyr Ile His Asp Asn Tyr Phe Leu Va - #l Asn Leu Val Asp Asn           610              - #   615              - #   620                           - - Asp Phe Pro Leu Asp Asn Cys Leu Trp Gln Va - #l Val Glu Asp Thr Leu       625                 6 - #30                 6 - #35                 6 -       #40                                                                               - - Glu Leu Leu Asn Arg Pro Thr Gln Asn Ala Ar - #g Glu Thr Glu Ala         Pro                                                                                              645  - #               650  - #               655              - -  - - (2) INFORMATION FOR SEQ ID NO: 5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 22 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: cDNA                                               - -    (iii) HYPOTHETICAL: YES                                                 - -     (iv) ANTI-SENSE: NO                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #5:                            - - AGCCTCAACC CCTGCTTGGA GG           - #                  - #                      22                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO: 6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 22 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: cDNA                                               - -    (iii) HYPOTHETICAL: YES                                                 - -     (iv) ANTI-SENSE: YES                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #6:                            - - TGACAGTTTG CTCCCCAGGC AC           - #                  - #                      22                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO: 7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 23 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: cDNA                                               - -    (iii) HYPOTHETICAL: YES                                                 - -     (iv) ANTI-SENSE: NO                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #7:                            - - TGAAGGAGAA GGTGTCTGCG GGA           - #                  - #                     23                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO: 8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 21 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: cDNA                                               - -    (iii) HYPOTHETICAL: YES                                                 - -     (iv) ANTI-SENSE: YES                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #8:                            - - AGGACGGTGC GGTGAGAGTG G           - #                  - #                       - #21                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO: 9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 23 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: cDNA                                               - -    (iii) HYPOTHETICAL: YES                                                 - -     (iv) ANTI-SENSE: NO                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #9:                            - - CACTGTGGTT GGCATGGATG ATG           - #                  - #                     23                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO: 10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 21 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: cDNA                                               - -    (iii) HYPOTHETICAL: YES                                                 - -     (iv) ANTI-SENSE: YES                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #10:                           - - GGCTGCTCTT GGACCCTCCT C           - #                  - #                       - #21                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO: 11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 21 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: cDNA                                               - -    (iii) HYPOTHETICAL: YES                                                 - -     (iv) ANTI-SENSE: NO                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #11:                           - - TGCTTCCGGC TCCCTCTAGC C           - #                  - #                       - #21                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO: 12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 22 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: cDNA                                               - -    (iii) HYPOTHETICAL: YES                                                 - -     (iv) ANTI-SENSE: YES                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #12:                           - - CCTCCCGCTC CCAAGAACAA AG           - #                  - #                      22                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO: 13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 23 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: cDNA                                               - -    (iii) HYPOTHETICAL: YES                                                 - -     (iv) ANTI-SENSE: NO                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #13:                           - - TGAAGGAGAA GGTGTCTGCG GGA           - #                  - #                     23                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO: 14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 20 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: cDNA                                               - -    (iii) HYPOTHETICAL: YES                                                 - -     (iv) ANTI-SENSE: NO                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #14:                           - - AGGACGGTGC GGTGAGAGTG            - #                  - #                       - # 20                                                                  __________________________________________________________________________ 

We claim:
 1. A cDNA probe for a human methylenetetrahydrofolate reductase (MTHFR) gene, said probe comprising a nucleotide sequence as set forth in SEQ ID NO:1 or a nucleoude sequence which encodes an amino acid sequence as set forth in SEQ ID NO:
 2. 2. A method of diagnosis of methylenetetrahydrofolate reductase (MTHFR) deficiency in a patient which comprises the steps of:a) amplifying a DNA sample from said patient or reverse-transcribing an RNA sample from said patient into DNA and amplifying said DNA; and b) analyzing the amplified DNA of step a) to determine whether said sample comprises at least one disease-causing sequence abnormality with respect to the human MTHFR nucleotide sequence as set forth in SEQ ID NO: 1 or a sequence encoding the human MTHFR amino acid sequence as set forth in SEQ ID NO: 2, said abnormality leading to a decrease in MTHFR activity and being indicative of MTHFR deficiency.
 3. The method of claim 2, wherein said MTHFR deficiency is associated with a disorder selected from the group consisting of cardiovascular disorders, cancer, neurological disorders, and disorders influenced by folic acid metabolism.
 4. The method of claim 2, wherein said sequence abnormality comprises a mutation selected from the group consisting of 167G→A, 482G→A, 559C→T, 677C→T, 692C→T, 764C→T, 792+1A→T, 985C→T, 1015C→T, and 1081C→T.
 5. The diagnosis method of claim 2, wherein said MTHFR deficiency is associated with an increased risk of occurrence of a neural tube defect in an offspring of said patient.
 6. The method of claim 3, wherein said cancer is selected from the group consisting of neuroblastomas and colorectal carcinomas.
 7. The method of diagnosis of claim 4, wherein said sequence abnormality consists of the 677C→T sequence abnormality. 